Dna molecules encoding l-glutamate-gated chloride channels from rhipicephalus sanguineus

ABSTRACT

The present invention relates in part to isolated nucleic acid molecules (polynucleotides) which encode  Rhipicephalus sanguineus  glutamate gated chloride channels. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding  R. sanguineus  glutamate gated chloride channels, substantially purified forms of associated  R. sanguineus  glutamate gated chloride channels and recombinant membrane fractions comprising these proteins, associated mutant proteins, and methods associated with identifying compounds which modulate associated  Rhipicephalus sanguineus  glutamate gated chloride channels, which will be useful as insecticides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. § 119(e),to provisional application U.S. Serial No. 60/193,934, filed Mar. 31,2000.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

[0002] Not Applicable

REFERENCE TO MICROFICHE APPENDIX

[0003] Not Applicable

FIELD OF THE INVENTION

[0004] The present invention relates in part to isolated nucleic acidmolecules (polynucleotides) which encode Rhipicephalus sanguineus (browndog tick) glutamate-gated chloride channels. The present invention alsorelates to recombinant vectors and recombinant hosts which contain a DNAfragment encoding R. sanguineus glutamate-gated chloride channels,substantially purified forms of associated R. sanguineus glutamate-gatedchloride channels and recombinant membrane fractions comprising theseproteins, associated mutant proteins, and methods associated withidentifying compounds which modulate associated Rhipicephalus sanguineusglutamate-gated chloride channels, which will be useful as insecticides.

BACKGROUND OF THE INVENTION

[0005] Glutamate-gated chloride channels, or H-receptors, have beenidentified in arthropod nerve and muscle (Lingle et al, 1981, Brain Res.212: 481-488; Horseman et al., 1988, Neurosci. Lett. 85: 65-70; Waffordand Sattelle, 1989, J. Exp. Bio. 144: 449-462; Lea and Usherwood, 1973,Comp. Gen. Parmacol. 4: 333-350; and Cull-Candy, 1976, J. Physiol. 255:449-464).

[0006] Invertebrate glutamate-gated chloride channels are importanttargets for the widely used avermectin class of anthelmintic andinsecticidal compounds. The avermectins are a family of macrocycliclactones originally isolated from the actinomycete Streptomycesavermitilis. The semisynthetic avermectin derivative, ivermectin(22,23-dihydro-avermectin B_(1a)), is used throughout the world to treatparasitic helminths and insect pests of man and animals. The avermectinsremain the most potent broad spectrum endectocides exhibiting lowtoxicity to the host. After many years of use in the field, thereremains little resistance to avermectin in the insect population. Thecombination of good therapeutic index and low resistance stronglysuggests that the glutamate-gated chloride (GluCl) channels remain goodtargets for insecticide development.

[0007] Glutamate-gated chloride channels have been cloned from the soilnematode Caenorhabditis elegans (Cully et al., 1994, Nature 371:707-711; see also U.S. Pat. No. 5,527,703 and Arena et al., 1992,Molecular Brain Research. 15: 339-348) and Ctenocephalides felis (flea;see WO 99/07828).

[0008] In addition, a gene encoding a glutamate-gated chloride channelfrom Drosophila melanogaster was previously identified (Cully et al.,1996, J. Biol. Chem. 271: 20187-20191; see also U.S. Pat. No.5,693,492).

[0009] Despite the identification of the aforementioned cDNA clonesencoding GluCl channels, it would be advantageous to identify additionalgenes which encode R. sanguineus GluCl channels in order to allow forimproved screening to identify novel GluCl channel modulators that mayhave insecticidal, acaricidal and/or nematocidal activity for animalhealth, especially as related to treatment of tick and mite infestationin dogs, cats, cattle, sheep and other agriculturally important animals.The present invention addresses and meets these needs by disclosingnovel genes which express a R. sanguineus GluCl1 and R. sanguineusGluCl2 channel wherein expression of these R. sanguineus GluCl RNAs inXenopus oocytes or other appropriate host cells result in an activeGluCl channel. Heterologous expression of a GluCl channel of the presentinvention will allow the pharmacological analysis of compounds activeagainst parasitic invertebrate species relevant to animal and humanhealth, especially in the treatment of tick infestations in dogs andcats. Heterologous cell lines expressing an active GluCl channel can beused to establish functional or binding assays to identify novel GluClchannel modulators that may be useful in control of the aforementionedspecies groups.

SUMMARY OF THE INVENTION

[0010] The present invention relates to an isolated or purified nucleicacid molecule (polynucleotide) which encodes a novel Rhipicephalussanguineus (brown dog tick) invertebrate GluCl1 channel protein. The DNAmolecules disclosed herein may be transfected into a host cell of choicewherein the recombinant host cell provides a source for substantiallevels of an expressed functional single, homomultimeric orheteromultimeric LGIC. Such functional ligand-gated ion channels maypossibly respond to other known ligands which will in turn provide foradditional screening targets to identify modulators of these channels,modulators which may act as effective insecticidal, mitacidal and/ornematocidal treatment for use in animal and human health and/or cropprotection.

[0011] The present invention relates to an isolated or purified nucleicacid molecule (polynucleotide) which encodes a novel Rhipicephalussanguineus invertebrate GluCl2 channel protein.

[0012] The present invention further relates to an isolated nucleic acidmolecule (polynucleotide) which encodes mRNA which expresses a novelRhipicephalus sanguineus GluCl1 channel protein, this DNA moleculecomprising the nucleotide sequence disclosed herein as SEQ ID NO:1, SEQID NO:3, and SEQ ID NO:5.

[0013] The present invention further relates to an isolated nucleic acidmolecule (polynucleotide) which encodes mRNA which expresses a novelRhipicephalus sanguineus GluCl2 channel protein, this DNA moleculecomprising the nucleotide sequence disclosed herein as SEQ ID NO:7.

[0014] The present invention also relates to biologically activefragments or mutants of SEQ ID NOs:1, 3, 5 and 7 which encodes mRNAexpressing a novel Rhipicephalus sanguineus invertebrate GluCl1 orGluCl2 channel protein, respectively. Any such biologically activefragment and/or mutant will encode either a protein or protein fragmentwhich at least substantially mimics the pharmacological properties of aR. sanguineus GluCl channel protein, including but not limited to the R.sanguineus GluCl1 channel proteins as set forth in SEQ ID NO:2, SEQ IDNO:4, and SEQ ID NO:6 as well as the respective GluCl2 channel proteinas set forth in SEQ ID NO:8. Any such polynucleotide includes but is notnecessarily limited to nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations such thatthese mutations encode mRNA which express a functional R. sanguineusGluCl channel in a eukaryotic cell, such as Xenopus oocytes, so as to beuseful for screening for agonists and/or antagonists of R. sanguineusGluCl activity.

[0015] A preferred aspect of this portion of the present invention isdisclosed in FIG. 1 (SEQ ID NO:1; designated T12), FIG. 3 (SEQ ID NO:3;designated T82) and FIG. 5 (SEQ ID NO:5; designated T32) encoding novelRhipicephalus sanguineus GluCl1 proteins, and FIG. 7 (SEQ ID NO:7,designated B1) encoding a novel Rhipicephalus sanguineus GluCl2 protein.

[0016] The isolated nucleic acid molecules of the present invention mayinclude a deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

[0017] The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which containthe substantially purified nucleic acid molecules disclosed throughoutthis specification.

[0018] The present invention also relates to a substantially purifiedform of an R. sanguineus GluCl1 channel protein, which comprises theamino acid sequence disclosed in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ IDNO:4) and FIG. 6 (SEQ ID NO:6), as well as to a novel Rhipicephalussanguineus GluCl2 protein, which comprises the amino acid sequencedisclosed in FIG. 8 (SEQ ID NO:8).

[0019] A preferred aspect of this portion of the present invention is aR. sanguineus GluCl1 channel protein which consists of the amino acidsequence disclosed in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) andFIG. 6 (SEQ ID NO:6).

[0020] Another preferred aspect of this portion of the present inventionis a R. sanguineus GluCl2 channel protein which consists of the aminoacid sequence disclosed in FIG. 8 (SEQ ID NO:8).

[0021] Another preferred aspect of the present invention relates to asubstantially purified, fully processed (including any proteolyticprocessing, glycosylation and/or phosphorylation) mature GluCl channelprotein obtained from a recombinant host cell containing a DNAexpression vector comprises a nucleotide sequence as set forth in SEQ IDNOs: 1, 3, 5 and/or 7 and expresses the respective RsGluCl1 or RsGluCl2precursor protein. It is especially preferred that the recombinant hostcell be a eukaryotic host cell, including but not limited to a mammaliancell line, an insect cell line such as an S2 cell line, or Xenopusoocytes.

[0022] Another preferred aspect of the present invention relates to asubstantially purified membrane preparation, partially purified membranepreparation, or cell lysate which has been obtained from a recombinanthost cell transformed or transfected with a DNA expression vector whichcomprises and appropriately expresses a complete open reading frame asset forth in SEQ ID NOs: 1, 3, 5 and/or 7, resulting in a functionalform of the respective RsGluCl1 or RsGluCl2 channel. The subcellularmembrane fractions and/or membrane-containing cell lysates from therecombinant host cells (both prokaryotic and eukaryotic as well as bothstably and transiently transformed or transfected cells) contain thefunctional proteins encoded by the nucleic acids of the presentinvention. This recombinant-based membrane preparation may comprise a R.sanguineus GluCl channel and is essentially free from contaminatingproteins, including but not limited to other R. sanguineus sourceproteins or host proteins from a recombinant cell which expresses theT12 (SEQ ID NO:2), T82 (SEQ ID NO:4) T32 (SEQ ID NO:6) GluCl1 channelprotein and/or the B1 (SEQ ID NO:8) GluCl2 channel protein. Therefore, apreferred aspect of the invention is a membrane preparation whichcontains a R. sanguineus GluCl channel comprising a GluCl proteincomprising the functional form of the full length GluCl1 channelproteins as disclosed in FIG. 2 (SEQ ID NO:2; T12), FIG. 4 (SEQ ID NO:4;T82), and FIG. 6 (SEQ ID NO:6, T32) and/or a functional form of the fulllength GluCl2 channel protein as disclosed in FIG. 8 (SEQ ID NO:8; B1).These subcellular membrane fractions will comprise either wild-type ormutant variations which are biologically functional forms of the R.sanguineus GluCl channels, any homomultimeric or heteromultimericcombination thereof (e.g. including but not

[0023] The present invention also relates to biologically activefragments and/or mutants of a R. sanguineus GluCl1 channel protein,comprising the amino acid sequence as set forth in SEQ ID NOs:2, 4and/or 6, as well as biologically active fragments and/or mutants of aR. sanguineus GluCl2 channel protein, comprising the amino acid sequenceas set forth in SEQ ID NO:8, including but not necessarily limited toamino acid substitutions, deletions, additions, amino terminaltruncations and carboxy-terminal truncations such that these mutationsprovide for proteins or protein fragments of diagnostic, therapeutic orprophylactic use and would be useful for screening for selectivemodulators, including but not limited to agonists and/or antagonists forR. sanguineus GluCl channel pharmacology.

[0024] A preferred aspect of the present invention is disclosed in FIG.2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6) and FIG. 8(SEQ ID NO:8), respective amino acid sequences which comprise the R.sanguineus GluCl1 and GluCl2 proteins of the present invention,respectively. Characterization of one or more of these channel proteinsallows for screening methods to identify novel GluCl channel modulatorsthat may have insecticidal, mitacidal and/or nematocidal activity foranimal health or crop protection. As noted above, heterologousexpression of a Rhipicephalus sanguineus GluCl channel will allow thepharmacological analysis of compounds active against parasiticinvertebrate species relevant to animal and human health, especiallydogs and cats, which are known to suffer from frequent tickinfestations. Heterologous cell lines expressing a functional RsGluCl1channel (e.g., functional forms of SEQ ID NOs:2, 4 and/or 6) or RsGluCl2channel (e.g., a functional form of SEQ ID NO:8), can be used toestablish functional or binding assays to identify novel GluCl channelmodulators that may be useful in control of the aforementioned speciesgroups.

[0025] The present invention also relates to polyclonal and monoclonalantibodies raised in response to the disclosed forms of RsGluCl1 and/orRsGluCl2, or a biologically active fragment thereof.

[0026] The present invention also relates to RsGluCl1 and/or RsGluCl2fusion constructs, including but not limited to fusion constructs whichexpress a portion of the RsGluCl linked to various markers, includingbut in no way limited to GFP (Green fluorescent protein), the MYCepitope, and GST. Any such fusion constructs may be expressed in thecell line of interest and used to screen for modulators of one or moreof the RsGluCl proteins disclosed herein.

[0027] The present invention relates to methods of expressing R.sanguineus GluCl1 and/or RsGluCl2 channel proteins and biologicalequivalents disclosed herein, assays employing these gene products,recombinant host cells which comprise DNA constructs which express theseproteins, and compounds identified through these assays which act asagonists or antagonists of GluCl channel activity.

[0028] It is an object of the present invention to provide an isolatednucleic acid molecule (e.g., SEQ ID NOs:1, 3, 5, and 7) which encodes anovel form of R. sanguineus GluCl, or fragments, mutants or derivativesRsGluCl1 or RsGluCl2, these proteins as set forth in SEQ ID NOs:2, 4, 6and 8, respectively. Any such polynucleotide includes but is notnecessarily limited to nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations such thatthese mutations encode mRNA which express a protein or protein fragmentof diagnostic, therapeutic or prophylactic use and would be useful forscreening for selective modulators for invertebrate GluCl pharmacology.

[0029] It is a further object of the present invention to provide the R.sanguineus GluCl proteins or protein fragments encoded by the nucleicacid molecules referred to in the preceding paragraph.

[0030] It is a further object of the present invention to providerecombinant vectors and recombinant host cells which comprise a nucleicacid sequence encoding R. sanguineus GluCl proteins or a biologicalequivalent thereof.

[0031] It is an object of the present invention to provide asubstantially purified form of R. sanguineus GluCl1 or GluCl2 proteins,respectively, as set forth in SEQ ID NOs:2, 4, 6, and 8.

[0032] It is another object of the present invention to provide asubstantially purified recombinant form of a R. sanguineus GluCl proteinwhich has been obtained from a recombinant host cell transformed ortransfected with a DNA expression vector which comprises andappropriately expresses a complete open reading frame as set forth inSEQ ID NOs: 1, 3, 5, and 7, resulting in a functional, processed form ofthe respective RsGluCl channel. It is especially preferred that therecombinant host cell be a eukaryotic host cell, such as a mammaliancell line.

[0033] It is an object of the present invention to provide forbiologically active fragments and/or mutants of R. sanguineus GluCl1 orGluCl2 proteins, respectively, such as set forth in SEQ ID NOs:2, 4, 6,and 8, including but not necessarily limited to amino acidsubstitutions, deletions, additions, amino terminal truncations andcarboxy-terminal truncations such that these mutations provide forproteins or protein fragments of diagnostic, therapeutic and/orprophylactic use.

[0034] It is further an object of the present invention to provide forsubstantially purified subcellular membrane preparation, partiallypurified membrane preparation or crude lysate from recombinant cellswhich comprise a pharmacologically active R. sanguineus GluCl1 orGluCl2-containing single, homomultimeric or heteromultimer channel,respectively, especially subcellular fractions obtained from a host celltransfected or transformed with a DNA vector comprising a nucleotidesequence which encodes a protein which comprises the amino acid as setforth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ IDNO:6), and FIG. 8 (SEQ ID NO:8).

[0035] It is another object of the present invention to provide asubstantially purified membrane preparation, partially purified membranepreparation, or crude lysate obtained from a recombinant host celltransformed or transfected with a DNA expression vector which comprisesand appropriately expresses a complete open reading frame as set forthin SEQ ID NOs: 1, 3, 5, and/or 7, resulting in a functional, processedform of the respective RsGluCl channel. It is especially preferred isthat the recombinant host cell be a eukaryotic host cell, including butnot limited to a mammalian cell line, an insect cell line such as an S2cell line, or Xenopus oocytes.

[0036] It is also an object of the present invention to use R.sanguineus GluCl proteins or membrane preparations containing R.sanguineus GluCl proteins or a biological equivalent to screen formodulators, preferably selective modulators, of R. sanguineus GluClchannel activity. Any such compound may be useful in screening for andselecting compounds active against parasitic invertebrate speciesrelevant to animal and human health. Such species include but are notlimited to worms, fleas, ticks, mites and lice. These membranepreparations may be generated from heterologous cell lines expressingthese GluCls and may constitute full length protein, biologically activefragments of the full length protein or may rely on fusion proteinsexpressed from various fusion constructs which may be constructed withmaterials available in the art.

[0037] As used herein, “substantially free from other nucleic acids”means at least 90%, preferably 95%, more preferably 99%, and even morepreferably 99.9%, free of other nucleic acids. As used interchangeablywith the terms “substantially free from other nucleic acids” or“substantially purified” or “isolated nucleic acid” or “purified nucleicacid” also refer to a DNA molecules which comprises a coding region fora R. sanguineus GluCl protein that has been purified away from othercellular components. Thus, a R. sanguineus GluCl DNA preparation that issubstantially free from other nucleic acids will contain, as a percentof its total nucleic acid, no more than 10%, preferably no more than 5%,more preferably no more than 1%, and even more preferably no more than0.1%, of non-R. sanguineus GluCl nucleic acids. Whether a given R.sanguineus GluCl DNA preparation is substantially free from othernucleic acids can be determined by such conventional techniques ofassessing nucleic acid purity as, e.g., agarose gel electrophoresiscombined with appropriate staining methods, e.g., ethidium bromidestaining, or by sequencing.

[0038] As used herein, “substantially free from other proteins” or“substantially purified” means at least 90%, preferably 95%, morepreferably 99%, and even more preferably 99.9%, free of other proteins.Thus, a R. sanguineus GluCl protein preparation that is substantiallyfree from other proteins will contain, as a percent of its totalprotein, no more than 10%, preferably no more than 5%, more preferablyno more than 1%, and even more preferably no more than 0.1%, of non-R.sanguineus GluCl proteins. Whether a given R. sanguineus GluCl proteinpreparation is substantially free from other proteins can be determinedby such conventional techniques of assessing protein purity as, e.g.,sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)combined with appropriate detection methods, e.g., silver staining orimmunoblotting. As used interchangeably with the terms “substantiallyfree from other proteins” or “substantially purified”, the terms“isolated R. sanguineus GluCl protein” or “purified R. sanguineus GluClprotein” also refer to R. sanguineus GluCl protein that has beenisolated from a natural source. Use of the term “isolated” or “purified”indicates that R. sanguineus GluCl protein has been removed from itsnormal cellular environment. Thus, an isolated R. sanguineus GluClprotein may be in a cell-free solution or placed in a different cellularenvironment from that in which it occurs naturally. The term isolateddoes not imply that an isolated R. sanguineus GluCl protein is the onlyprotein present, but instead means that an isolated R. sanguineus GluClprotein is substantially free of other proteins and non-amino acidmaterial (e.g., nucleic acids, lipids, carbohydrates) naturallyassociated with the R. sanguineus GluCl protein in vivo. Thus, a R.sanguineus GluCl protein that is recombinantly expressed in aprokaryotic or eukaryotic cell and substantially purified from this hostcell which does not naturally (i.e., without intervention) express thisGluCl protein is of course “isolated R. sanguineus GluCl protein” underany circumstances referred to herein. As noted above, a R. sanguineusGluCl protein preparation that is an isolated or purified R. sanguineusGluCl protein will be substantially free from other proteins willcontain, as a percent of its total protein, no more than 10%, preferablyno more than 5%, more preferably no more than 1%, and even morepreferably no more than 0.1%, of non-R. sanguineus GluCl proteins.

[0039] As used interchangeably herein, “functional equivalent” or“biologically active equivalent” means a protein which does not haveexactly the same amino acid sequence as naturally occurring R.sanguineus GluCl, due to alternative splicing, deletions, mutations,substitutions, or additions, but retains substantially the samebiological activity as R. sanguineus GluCl. Such functional equivalentswill have significant amino acid sequence identity with naturallyoccurring R. sanguineus GluCl and genes and cDNA encoding suchfunctional equivalents can be detected by reduced stringencyhybridization with a DNA sequence encoding naturally occurring R.sanguineus GluCl. For example, a naturally occurring R. sanguineusGluCl1 protein disclosed herein comprises the amino acid sequence shownas SEQ ID NO:2 and is encoded by SEQ ID NO:1. A nucleic acid encoding afunctional equivalent has at least about 50% identity at the nucleotidelevel to SEQ ID NO:1.

[0040] As used herein, “a conservative amino acid substitution” refersto the replacement of one amino acid residue by another, chemicallysimilar, amino acid residue. Examples of such conservative substitutionsare: substitution of one hydrophobic residue (isoleucine, leucine,valine, or methionine) for another; substitution of one polar residuefor another polar residue of the same charge (e.g., arginine for lysine;glutamic acid for aspartic acid).

[0041] As used herein, “LGIC” refers to a—ligand-gated ion channel—.

[0042] As used herein, “GluCl” refers to—L-glutamate gated chloridechannel—.

[0043] As used herein, “RsGluCl” refers to—Rhipicephalus sanguineusL-glutamate gated chloride channel—.

[0044] Furthermore, as used herein “RsGluCl” may refer to RsGluCl1and/or RsGluCl2.

[0045] As used herein, the term “mammalian” will refer to any mammal,including a human being.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046]FIG. 1 shows the nucleotide sequence of the R. sanguineus GluCl1cDNA clone, T12, set forth in SEQ ID NO:1.

[0047]FIG. 2 shows the amino acid sequence of the R. sanguineus GluCl1protein, T12, as set forth in SEQ ID NO:2.

[0048]FIG. 3 shows the nucleotide sequence of the R. sanguineus GluCl1cDNA clone, T82, as set forth in SEQ ID NO:3.

[0049]FIG. 4 shows the amino acid sequence of the R. sanguineus GluCl1protein, T82, as set forth in SEQ ID NO:4.

[0050]FIG. 5 shows the nucleotide sequence of the R. sanguineus GluCl1cDNA clone, T32, as set forth in SEQ ID NO:5.

[0051]FIG. 6 shows the amino acid sequence of the R. sanguineus GluCl1protein, T32, as set forth in SEQ ID NO:6.

[0052]FIG. 7 shows the nucleotide sequence of the R. sanguineus GluCl2cDNA clone, B1, as set forth in SEQ ID NO:7.

[0053]FIG. 8 shows the amino acid sequence of the R. sanguineus GluCl2protein, B1, as set forth in SEQ ID NO:8.

[0054]FIG. 9 shows the amino acid sequence comparison for RsGluCl1 [T12(SEQ ID NO:2), T82 (SEQ ID NO:4), T32 (SEQ ID NO:6) and RsGluCl2 (B1,SEQ ID NO:8) proteins.

[0055]FIG. 10 shows the glutamate-activated current in Xenopus oocytesinjected with RsGluCl1 T12 RNA. Current activation was maximal with 10μM glutamate. and no current was seen in uninjected oocytes.

[0056]FIG. 11 shows the activation by ivermectin of RsGluCl2 expressedin Xenopus oocytes. Current activation was maximal with ˜1 μMivermectin.

DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention relates to an isolated nucleic acidmolecule (polynucleotide) which encodes a Rhipicephalus sanguineusinvertebrate GluCl channel protein. The isolated or purified nucleicacid molecules of the present invention are substantially free fromother nucleic acids. For most cloning purposes, DNA is a preferrednucleic acid. As noted above, the DNA molecules disclosed herein may betransfected into a host cell of choice wherein the recombinant host cellprovides a source for substantial levels of an expressed functionalsingle, homomultimeric or heteromultimeric GluCl channel. Suchfunctional ligand-gated ion channels may possibly respond to other knownligands which will in turn provide for additional screening targets toidentify modulators of these channels, modulators which may act aseffective insecticidal, mitacidal and/or nematocidal treatment for usein animal and human health and/or crop protection. It is shown hereinthat RsGluCl 1 exhibits a current in response to glutamate and that anRsGluCl2 channel protein expressed in Xenopus oocytes exhibit a currentin response to the addition of ivermectin phosphate. However, it shouldbe noted that a single channel subunit protein might not form afunctional channel, such as seen with the GABA-A subunit gamma, whichdoes not express a functional homomultimer. Therefore, the expressedproteins of the present invention may function in vivo as a component ofa wild type ligand-gated ion channel which contains a number ofaccessory and/or channel proteins, including the channel proteinsdisclosed herein. However, the GluCl proteins of the present inventionneed not directly mimic the wild type channel in order to be useful tothe skilled artisan. Instead, the ability to form a functional, single,membrane associated channel within a recombinant host cell renders theseproteins amenable to the screening methodology known in the art anddescribed in part within this specification. Therefore, as noted withinthis specification, the disclosed Rs channel proteins of the presentinvention are useful as single functional channels, as a homomultimericchannel or as a heteromultimeric channel with various proteins disclosedherein with or without additional Rs channel subunit proteins oraccessory proteins which may contribute to the full, functional GluClchannel. As noted above, the DNA molecules disclosed herein may betransfected into a host cell of choice wherein the recombinant host cellprovides a source for substantial levels of an expressed functionalsingle, homomultimeric or heteromultimeric GluCl. Such functionalligand-gated ion channels may possibly respond to other known ligandswhich will in turn provide for additional screening targets to identifymodulators of these channels, modulators which may act as effectiveinsecticidal, mitacidal and/or nematocidal treatment for use in animaland human health and/or crop protection

[0058] The present invention relates to an isolated nucleic acidmolecule (polynucleotide) which encodes mRNA which expresses a novelRhipicephalus sanguineus invertebrate GluCl1 channel protein, this DNAmolecule comprising the nucleotide sequence disclosed herein as SEQ IDNO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0059] The present invention relates to an isolated nucleic acidmolecule polynucleotide) which encodes mRNA which expresses a novelRhipicephalus sanguineus invertebrate GluCl2 channel protein, this DNAmolecule comprising the nucleotide sequence disclosed herein as SEQ IDNO:7.

[0060] The isolation and characterization of the RsGluCl nucleic acidmolecules of the present invention were identified as described indetail in Example Section 1. These cDNA molecules, as discussed herein,are especially useful to establish novel insecticide screens, validatepotential lead compounds with insecticidal activity, especially for usein treating cattle, dog and cat tick and mite infestations or that maykill other arachnids, and use these novel cDNA sequences ashybridization probes to isolate related genes from other organisms toestablish additional pesticide drug screens. The RsGluCl1 and RsGluCl2encoding cDNAs of the present invention were isolated from the brown dogtick species Rhipicephalus sanguineus. The DNA sequence predictsproteins that share common features with the class of chloride channelssensitive to glutamate and ivermectin. When the RsGluCl1 or RsGluCl2cDNAs are expressed in Xenopus oocytes, a glutamate andivermectin-sensitive channel is observed. The pharmacology of compoundsthat act at these channels would likely be different between thesespecies. By screening on the arachnid channel it will be more likely todiscover arachnid-specific compounds. Therefore, the cDNAs of thepresent invention can be expressed in cell lines or other expressionsystems and used for competition binding experiments or for functionalchloride channel assays to screen for compounds that activate, block ormodulate the channel.

[0061] Invertebrate glutamate-gated chloride channels (GluCls) arerelated to the glycine- and GABA-gated chloride channels and aredistinct from the excitatory glutamate receptors (e.g. NMDA or AMPAreceptors). The first two members of the GluCl family were identified inthe nematode C. elegans, following a functional screen for the receptorof the anthelmintic drug ivermectin. Several additional GluCls have nowbeen cloned in other invertebrate species. However, there is no evidenceyet for GluCl counterparts in vertebrates; because of this, GluCls areexcellent targets for anthelmintics, insecticides, acaricides, etc.Specific GluCl modulators, such as nodulisporic acid and its derivativeshave an ideal safety profile because they lack mechanism-based toxicityin vertebrates. The present invention relates in part to three novel R.sanguineus GluCl1 clones, T12, T82 and T32, and a R. sanguineus GluCl2clone, B1. The RsGluCl1 cDNAs were isolated by low stringencyhybridization using a Drosophila GluCl probe representing the putativemembrane spanning domains, M1, M2 and M3. The RsGluCl2 cDNA was isolatedby PCR using degenerate primers representing conserved regions in amino-and the M2-domains of the GluCl proteins of Drosophila, flea (C. felis),and C. elegans. It appears that RNA editing (A to G transitions) occurin these cDNAs and have resulted in some amino acid changes.RsGluCl1-T12 and T82 are similar except for one amino acid differencewhile RsGluCl1-T32 contains two additional exons in the coding region.

[0062] The present invention relates to the isolated or purified DNAmolecule described in FIG. 1 (T12) and set forth as SEQ ID NO:1, whichencodes the R. sanguineus GluCl1 protein described in FIG. 2 and setforth as SEQ ID NO:2, the nucleotide sequence of T12 is as follows: (SEQID NO:1) 1 CGCTCCCCCA ATCCTGAGGT TCCTTCTAAC GAGAAGGAGG AGCCACAGCGCCGGCTGCGG 61 TACCGCCGCA CGGGCCAACG TGAGACCGCC CGAGCCCGGC GCCCTGACTTAGGCCGCTGA 121 GCGAAACCCA AGGCGGCGCG CTGGCCACTC CACGGGAACG AGACCGGCCCCCTGGAGACG 181 ACATCGTCGA CCACAATGAA CTACTTCTCT GACGTGGCGA AGATGGTGGCTTCATCGAAG 241 AGAGAAATCA TCGAAGCTTT CCACGCGACA TCTGGAGTAC ACGGCGCATGCGAATGAGCG 301 AACATCGCTG ACCGAGACTC GCCCGTCACC ATGAGCGTAC ATTCATGGCGCTTTTGTGTC 361 CCACTGGTGG CTCTAGCGTT TTTCTTGTTG ATTCTTCTGT CGTGTCCATCGGCATGGGGC 421 AAGGCAAATT TCCGCGCTAT AGAAAAGCGG ATATTGGACA GCATCATTGGCCAGGGTCGT 481 TATGACTGCA GGATCCGGCC CATGGGAATT AACAACACAG ACGGGCCGGCTCTTGTACGC 541 GTTAACATCT TTGTAAGAAG TATCGGCAGA ATTGATGACG TCACCATGGAGTACACAGTG 601 CAAATGACGT TCAGAGAGCA GTGGCGGGAC GAGAGACTCC AGTACGACGACTTGGGCGGC 661 CAGGTTCGCT ACCTGACGCT CACCGAACCG GACAAGCTTT GGAAGCCGGACCTGTTTTTC 721 TCCAACGAGA AAGAGGGACA CTTCCACAAC ATCATCATGC CCAACGTGCTTCTACGCATA 781 CATCCCAACG GCGACGTTCT CTTCAGCATC AGAATATCCT TGGTGCTTTCATGTCCGATG 841 AACCTGAAAT TTTATCCTTT GGATAAACAA ATCTGCTCTA TCGTCATGGTGAGCTATGGG 901 TATACAACAG AGGACCTGGT GTTTCTATGG AAAGAGGGGG ATCCTGTACAGGTCACAAAA 961 AATCTCCACT TGCCACGTTT CACGCTGGAA AGGTTTCAAA CCGACTACTGCACCAGTCGG 1021 ACCAACACTG GCGAGTACAG CTGCTTGCGC GTGGACCTGG TGTTCAAGCGCGAGTTCAGC 1081 TACTACCTGA TCCAGATCTA CATCCCGTGC TGCATGCTGG TCATCGTGTCCTGGGTGTCG 1141 TTCTGGCTCG ACCCCACCTC GATCCCGGCG CGAGTGTCGC TGGGCGTCACCACCCTGCTC 1201 ACCATGGCCA CGCAGATATC GGGCATCAAC GCCTCGCTGC CTCCCGTTTCCTACACCAAG 1261 GCCATTGACG TGTGGACCGG CGTCTGTCTG ACCTTCGTAT TCGGCGCGCTCCTCGAGTTC 1321 GCCCTGGTCA ACTACGCCTC GCGGTCAGAT TCACGCCGGC AGAACATGCAGAAGCAGAAG 1381 CAGAGGAAAT GGGAGCTCGA GCCGCCCCTG GACTCGGACC ACCTGGAGGACGGCGCCACC 1441 ACGTTCGCCA TGAGGCCGCT GGTGCACCAC CACGGAGAGC TGCATGCCGACAAGTTGCGG 1501 CAGTGCGAAG TCCACATGAA GACCCCCAAG ACGAACCTTT GCAAGGCCTGGCTTTCCAGG 1561 TTTCCCACGC GATCCAAACG CATCGACGTC GTCTCGCGGA TCTTCTTTCCGCTCATGTTC 1621 GCCCTCTTCA ACCTCGTCTA CTGGACAACC TACCTCTTCC GGGAAGACGAGGAAGACGAG 1681 TGACAGAACA CGGACGCCAC GACAGCCGCC ATCCGACACC ATCGTCACTGCAGGCACGCA 1741 CTCTGTCGCG CGCACACACC ACGAAGACCG GCGCGCCAAC GCACGATGCGCGTTGGCCGC 1801 TGAAAAACCC GGGAGCGGGG CGGTGGGGGA GGCTATGCCC CGGCCCCTCGCTCCTCATCC 1861 TCCGTGCACG CTCGAATCGT CATCGCCACA GCCAGAAAAA AAAAAGATACCGTGCGAAAA 1921 GTGGCGGCAA CACAACGTCG ACGCCATCAG CGCCGCCCAG AGCTGCAAGCGGCTCCCACA 1981 TGGTTGCCAC CGCAGCTTCC TCTACGACCC TTCATCCCCA CCGGCACCAGCTACGAGAAA 2041 GGGACCTTAT TTCGGGCCAT CCCTACATAG GCGACTGTTG TTTTCGCACGAAAGATCTTT 2101 ACGCAGCTGA TGCTGAAAAA AAAAAAAAAA AAAAAAAA.

[0063] The present invention also relates to the isolated or purifiedDNA molecule described in FIG. 3 (T82) and set forth as SEQ ID NO:3,which encodes the R. sanguineus GluCl1 protein described in FIG. 4 andset forth as SEQ ID NO:4, the nucleotide sequence T82 as follows: (SEQID NO:3) 1 CACACCTCCT GCGTCTCTCC ACTCGATGAA GACCTGTCCC GGAGGCGCGAGCCCAACTGC 61 GCGCTCTGTC CGCATGTGTC GCCGCCACTG AGAGGCCTCC GGCGTGGCGCGCTTGTCACC 121 GCGGCGCGCC GGCCCGCAGC AAATCGCGGG CATTCCACTC AGGGTCTCATTCGCTCCCCC 181 AATCCTGAGG TTCCTTCTAA CGAGAAGGAG GAGCCACAGC GCCGGCTGCGGTACCGCCGC 241 ACGGGCCAAC GTGAGACCGC CCGAGCCCGG CGCCCTGACT TAGGCCGCTGAGCGAAACCC 301 AAGGCGGCGC GCTGGCCACT CCACGGGAAC GAGACCGGCC CCCTGGAGACGACATCGTCG 361 ACCACAATGA ACTACTTCTC TGACGTGGCG AAGATGGTGG CTTCATCGAAGAGAGAAATC 421 ATCGAAGCTT TCCACGCGAC ATCTGGAGTA CACGGCGCAT GCGAATGAGCGAACATCGCT 481 GACCGAGACT CGCCCGTCAC CATGAGCGTA CATTCATGGC GCTTTTGTGTCCCACTGGTG 541 GCTCTAGCGT TTTTCTTGTT GATTCTTCTG TCGTGTCCAT CGGCATGGGGCAAGGCAAAT 601 TTCCGCGCTA TAGAAAAGCG GATATTGGAC AGCATCATTG GCCAGGGTCGTTATGACTGC 661 AGGATCCGGC CCATGGGAAT TAACAACACA GACGGGCCGG CTCTTGTACGCGTTAACATC 721 TTTGTAAGAA GTATCGGCAG AATTGATGAC GTCACCATGG AGTACACAGTGCAAATGACG 781 TTCAGAGAGC AGTGGCGGGA CGAGAGACTC CAGTACGACG ACTTGGGCGGCCAGGTTCGC 841 TACCTGACGC TCACCGAACC GGACAAGCTT TGGAAGCCGG ACCTGTTTTTCTCCAACGAG 901 AAAGAGGGAC ACTTCCACAA CATCATCATG CCCAACGTGC TTCTACGCATACATCCCAAC 961 GGCGACGTTC TCTTCAGCAT CAGAATATCC TTGGTGCTTT CATGTCCGATGAACCTGAAA 1021 TTTTATCCTT TGGATAAACA AATCTGCTCT ATCGTCATGG TGAGCTATGGGTATACAACA 1081 GAGGACCTGG TGTTTCTATG GAAAGAGGGG GATCCTGTAC AGGTCACAAAAAATCTCCAC 1141 TTGCCACGTT TCACGCTGGA AAGGTTTCAA ACCGACTACT GCACCAGTCGGACCAACACT 1201 GGCGAGTACA GCTGCTTGCG CGTGGACCTG GTGTTCAAGC GCGAGTTCAGCTACTACCTG 1261 ATCCAGATCT ACATCCCGTG CTGCATGCTG GTCATCGTGT CCTGGGTGTCGTTCTGGCTC 1321 GACCCCACCT CGATCCCGGC GCGAGTGTCG CTGGGCGTCA CCACCCTGCTCACCATGGCC 1381 ACGCAGATAT CGGGCATCAA CGCCTCGCTG CCTCCCGTTT CCTACACCAAGGCCATTGAC 1441 GTGTGGACCG GCGTCTGTCT GACCTTCGTA TTCGGCGCGC TCCTCGAGTTCGCCCTGGTC 1501 AACTACGCCT CGCGGTCAGA TTCACGCCGG CAGAACATGC AGAAGCAGAAGCAGAGGAAA 1561 TGGGAGCTCG AGCCGCCCCT GGACTCGGAC CACCTGGAGG ACGGCGCCACCACGTTCGCC 1621 ATGAGGCCGC TGGTGCACCA CCACGGAGAG CTGCATGCCG ACAAGTTGCGGCAGTGCGAA 1681 GTCCACATGA AGACCCCCAA GACGAACCTT TGCAAGGCCT GGCTTTCCAGGTTTCCCACG 1741 CGATCCCAAC GCATCGACGT CGTCTCGCGG ATCTTCTTTC CGCTCATGTTCGCCCTCTTC 1801 AACCTCGTCT ACTGGACAAC CTACCTCTTC CGGGAAGACA AGGAAGACGAGTGACAGAAC 1861 ACGAACGCCA CGACAGCCGC CATCCGACAC CATCGTCACT GCAGGCACGCACTCTGTCGC 1921 GCGCACACAC CACGAAGACC GGCGCGCCAA CGCACGATGC GCGTTGGCCGCTGAAAAACC 1981 CGGGAGCGGG GCGGTGGGGG AGGCTATGCC CCGGCCCCTC GCTCCTCATCCTCCGTGCAC 2041 GCTCGAATCG TCATCGCCAC AGCCAGAAAA AAAAAAGATA CCGTGCGAAAAGTGGCGGCA 2101 ACACAACGTC GACGCCATCA GCGCCGCCCA GAGCTGCAAG CGGCTCCCACATGGTTGCCA 2161 CCGCAGCTTC CTCTACGACC CTTCATCCCC ACCGGCACCA GCTACGAGAAAGGGACCTTA 2221 TTTCGGGCCA TCCCTACATA GGCGACTGTT GTTTTCGCAC GAAAGATCTTTACGCAGCTG 2281 ATGCTGAAA.

[0064] The present invention also relates to the isolated or purifiedDNA molecule described in FIG. 5 (T32) and set forth as SEQ ID NO:5,which encodes the R. sanguineus GluCl1 protein described in FIG. 6 andset forth as SEQ ID NO:6, the nucleotide sequence T32 as follows: (SEQID NO:5) 1 CAGGCTCCGG CGTGACTGTC GCTCGCTCGG CTCTCGACGC TCGCGGCGGGAACAACCGCT 61 ACCCGGACGC TCGATCAGGA GCAGTTCGGG CCACAGAGAA AGGGGCCGAGGAGTGCACAC 121 CTCCTGCGTC TCTCCACTCG ATGAAGACCT GTCCCGGAGG CGCGAGCCCAACTGCGCGCT 181 CTGTCCGCAT GTGTCGCCGC CACTGAGAGG CCTCCGGCGT GGCGCGCTTGTCAACGCGGC 241 GCGCCGGCCC GCAGCAAATC GCGGGCATTC CACTCAGGGT CTCATTCGCTCCCCCAATCC 301 TGAGGTTCCT TCTAACGAGA AGGAGGAGCC ACAGCGCCGG CTGCGGTACCGCCGCACGGG 361 CCAACGTGAG ACCGCCCGAG CCCGGCGCCC TGACTTAGGC CGCTGAGCGAAACCCAAGGC 421 GGCGCGCTGG CCACTCCACG GGAACGAGAC CGGCCCCCTG GAGACGACATCGTCGACCAC 481 AATGAACTAC TTCTCTGACG TGGCGAAGAT GGTGGCTTCA TCGAAGAGAGAAATCATCGA 541 AGCTTTCCAC GCGACATCTG GAGTACACGG CGCATGCGAA TGAGCGAACATCGCTGACCG 601 AGACTCGCCC GTCACCATGA GCGTACATTC ATGGCGCTTT TGTGTCCCACTGGTGGCTCT 661 AGCGTTTTTC TTGTTGATTC TTCTGTCGTG TCCATCGGCA TGGGCCGAAACGCTGCCTAC 721 GCCACCAACC CGTGGCCAGG GGGGCGTTCC GGTCGCGGCC GCGATGCTCCTGGGGAAACA 781 GCAAAGTTCC CGCTACCAAG ATAAAGAGGG CAAGGCAAAT TTCCGCGCTATAGAAAAGCG 841 GATATTGGAC AGCATCATTG GCCAGGGTCG TTATGACTGC AGGATCCGGCCCATGGGAAT 901 TAACAACACA GACGGGCCGG CTCTTGTACG CGTTAACATC TTTGTAAGAAGTATCGGCAG 961 AATTGATGAC GTCACCATGG AGTACACAGT GCAAATGACG TTCAGAGAGCAGTGGCGGGA 1021 CGAGAGACTC CAGTACGACG ACTTGGGCGG CCAGGTTCGC TACCTGACGCTCACCGAACC 1081 GGACAAGCTT TGGAAGCCGG ACCTGTTTTT CTCCAACGAG AAAGAGGGACACTTCCACAA 1141 CATCATCATG CCCAACGTGC TTCTACGCAT ACATCCCAAC GGCGACGTTCTCTTCAGCAT 1201 CAGAATATCC TTGGTGCTTT CATGTCCGAT GAACCTGAAA TTTTATCCTTTGGATAAACA 1261 AATCTGCTCT ATCGTCATGG TGAGCTATGG GTATACAACA GAGGACCTGGTGTTTCTATG 1321 GAAAGAGGGG GATCCTGTAC AGGTCACAAA AAATCTCCAC TTGCCACGTTTCACGCTGGA 1381 AAGGTTTCAA ACCGACTACT GCACCAGTCG GACCAACACT GGCGAGTACAGCTGCTTGCG 1441 CGTGGACCTG GTGTTCAAGC GCGAGTTCAG CTACTACCTG ATCCAGATCTACATCCCGTG 1501 CTGCATGCTG GTCATCGTGT CCTGGGTGTC GTTCTGGCTC GACCCCACCTCGATCCCGGC 1561 GCGAGTGTCG CTGGGCGTCA CCACCCTGCT CACCATGGCC ACGCAGATATCGGGCATCAA 1621 CGCCTCGCTG CCTCCCGTTT CCTACACCAA GGCCATTGAC GTGTGGACCGGCGTCTGTCT 1681 GACCTTCGTA TTCGGCGCGC TCCTCGAGTT CGCCCTGGTC AACTACGCCTCGCGGTCAGA 1741 TTCACGCCGG CAGAACATGC AGAAGCAGAA GCAGAGGAAA TGGGAGCTCGAGCCGCCCCT 1801 GGACTCGGAC CACCTGGAGG ACGGCGCCAC CACGTTCGCC ATGGTGAGCTCCGGCGAGCC 1861 GGCGGGCCTC ATGGCGCGAA CCTGGCCACC ACCGCCGCTG CCGCCAAACATGGCGGCCGG 1921 CTCCGCGCAA GCCGGCGCCA GGCCGCTGGT GCACCACCAC GGAGAGCTGCATGCCGACAA 1981 GTTGCGGCAG TGCGAAGTCC ACATGAAGAC CCCCAAGACG AACCTTTGCAAGGCCTGGCT 2041 TTCCAGGTTT CCCACGCGAT CCAAACGCAT CGACGTCGTC TCGCGGATCTTCTTTCCGCT 2101 CGTGTTCGCC CTCTTCAACC TCGTCTACTG GACAACCTAC CTCTTCCGGGAAGACGAGGA 2161 GGACGAGTGA CAGAACACGA ACGCCACGAC AGCCGCCATC CGACACCATCGTCACTGCAG 2221 GCACGCACTC TGTCGCGCGC ACACACCACG AAGACCGGCG CGCCAACGCACGATGCGCGT 2281 TGGCCGCTGA AAAACCCGGG AGCGGGGCGG TGGGGGAGGC TATGCCCCGGCCCCTCGCTC 2341 CTCATCCTCC GTGCACGCTC GAATCGTCAT CGCCACAGCC AGAAAAAAAAAAAAAAAAAA.

[0065] The present invention also relates to an isolated or purified DNAmolecule which encodes a R. sanguineus GluCl2 protein. One such nucleicacid is described in FIG. 7 (B1) and set forth as SEQ ID NO:7, whichencodes the R. sanguineus GluCl2 protein described in FIG. 8 and setforth as SEQ ID NO:8, the nucleotide sequence B1 as follows: 1CGCCGCTCAA TCGCGGGCTA CGGACTCGTC GTTCCCGGAG GGGCTTGGAC (SEQ ID NO:7) 51CACAGCTCGC TCGTCACCGT GGTGGCTGGC CGCTTCGCCT GGCGGTCCTG 101 CACGCACGCTGTAACGAACG TCGCCACGCG ATGTTTGGTG TGCCATGCTC 151 CCGCGCCTGC CGCCTTGTGGTGGTGATAGC TGCGTTCTGC TGGCCGCCCG 201 CTCTGCCGCT CGTACCCGGG GGAGTTTCCTCCAGAGCAAA CGATCTGGAC 251 ATTCTGGACG AGCTCCTCAA AAACTACGAT CGAAGGGCCCTGCCGAGCAG 301 TCACCTCGGA AATGCAACTA TTGTGTCATG CGAAATTTAC ATACGAAGTT351 TTGGATCAAT AAATCCTTCG AACATGGACT ACGAAGTCGA CCTCTACTTC 401CGGCAGTCGT GGCTCGACGA GCGGTTACGC AAATCCACGC TATCTCGTCC 451 GCTCGACCTTAATGACCCAA AGCTGGTACA AATGATATGG AAGCCAGAAG 501 TTTTCTTTGC GAACGCGAAACACGCCGAGT TCCAATATGT GACTGTACCT 551 AACGTCCTCG TTAGGATCAA CCCGACTGGAATAATCTTGT ACATGTTGCG 601 GTTAAAACTG AGGTTCTCCT GCATGATGGA CCTGTACCGGTACCCCATGG 651 ATTCCCAAGT CTGCAGCATC GAAATTGCCT CTTTTTCCAA AACCACCGAA701 GAGCTGCTGC TGAAATGGTC CGAGAGTCAG CCTGTCGTTC TCTTCGATAA 751CCTCAAGTTG CCCCAGTTTG AAATAGAGAA GGTGAACACG TCCTTATGCA 801 AAGAAAAGTTTCACATAGGG GAATACAGTT GCCTGAAAGC CGACTTCTAT 851 CTGCAGCGTT CCCTCGGTTATCACATGGTG CAGACCTATC TTCCGACCAC 901 GCTTATCGTG GTCATCTCAT GGGTGTCATTCTGGCTCGAC GTAGACGCCA 951 TACCCGCCCG TGTCACCCTG GGCGTAACCA CGCTGCTCACCATCTCATCC 1001 AAGGGTGCCG GTATCCAGGG AAACCTGCCT CCCGTCTCGT ACATCAAGGC1051 CATGGACGTC TGGATAGGAT CCTGTACTTC GTTTGTCTTT GCGGCCCTTC 1101TAGAGTTCAC ATTCGTCAAC TATCTCTGGA GGCGGCTGCC CAATAAGCGC 1151 CCATCTTCTGACGTACCGGT GACGAATATA CCAAGCGACG GCTCAAAGCA 1201 TGACATTGCG GCACAGCTCGTACTCGACAA GAATGGACAC ACCGAAGTTC 1251 GCACGTTGGT CCAAGCGATG CCACGCAGCGTCGGAAAAGT GAAGGCCAAG 1301 CAGATTGATC AACTCAGCCG AGTCGCCTTT CCCGCTCTTTTTCTCCTCTT 1351 CAACCTCGTG TACTGGCCGT ACTACATTAA GTCATAAAGA ACGTAGTTTT1401 CT.

[0066] The above-exemplified isolated DNA molecules, shown in FIGS. 1, 35, and 7, respectively, comprise the following characteristics:

[0067] T12 (SEQ ID NO: 1):

[0068] 2138 nuc.:initiating Met (nuc. 331-333) and “TGA” term. codon(nuc.1681-1683), the open reading frame resulting in an expressedprotein of 450 amino acids, as set forth in SEQ ID NO:2.

[0069] T82 (SEQ ID NO:3):

[0070] 2289 nuc.:initiating Met (nuc. 502-504) and “TGA” term. codon(nuc. 1852-1854), the open reading frame resulting in an expressedprotein of 450 amino acids, as set forth in SEQ ID NO:4.

[0071] T32 (SEQ ID NO:5):

[0072] 2400 nuc.:initiating Met (nuc. 617-619) and “TGA” term. codon(nuc. 2168-2170), the open reading frame resulting in an expressedprotein of 517 amino acids, as set forth in SEQ ID NO:6.

[0073] B1 (SEQ ID NO:7):

[0074] 1402 nuc.:initiating Met (nuc. 131-133) and “TAA” term. codon(nuc. 1385-1387), the open reading frame resulting in an expressedprotein of 418 amino acids, as set forth in SEQ ID NO:8.

[0075] The present invention also relates to biologically activefragments or mutants of SEQ ID NOs:1, 3, 5 and 7 which encodes mRNAexpressing a novel Rhipicephalus sanguineus invertebrate GluCl1 orGluCl2 channel protein, respectively. Any such biologically activefragment and/or mutant will encode either a protein or protein fragmentwhich at least substantially mimics the pharmacological properties of aR. sanguineus GluCl channel protein, including but not limited to the R.sanguineus GluCl1 channel proteins as set forth in SEQ ID NO:2, SEQ IDNO:4, and SEQ ID NO:6 as well as the respective GluCl2 channel proteinas set forth in SEQ ID NO:8. Any such polynucleotide includes but is notnecessarily limited to nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations such thatthese mutations encode mRNA which express a functional R. sanguineusGluCl channel in a eukaryotic cell, such as Xenopus oocytes, so as to beuseful for screening for agonists and/or antagonists of R. sanguineusGluCl activity.

[0076] A preferred aspect of this portion of the present invention isdisclosed in FIG. 1 (SEQ ID NO:1; designated T12), FIG. 3 (SEQ ID NO:3;designated T82) and FIG. 5 (SEQ ID NO:5; designated T32) encoding novelRhipicephalus sanguineus GluCl1 proteins, and FIG. 7 (SEQ ID NO:7,designated B1) encoding a novel Rhipicephalus sanguineus GluCl2 protein.

[0077] The isolated nucleic acid molecules of the present invention mayinclude a deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

[0078] The degeneracy of the genetic code is such that, for all but twoamino acids, more than a single codon encodes a particular amino acid.This allows for the construction of synthetic DNA that encodes theRsGluCl1 or RsGluCl2 protein where the nucleotide sequence of thesynthetic DNA differs significantly from the nucleotide sequence of SEQID NOs:1, 3, 5, and 7 but still encodes the same RsGluCl protein as SEQID NO:1, 3, 5 and 7. Such synthetic DNAs are intended to be within thescope of the present invention. If it is desired to express suchsynthetic DNAs in a particular host cell or organism, the codon usage ofsuch synthetic DNAs can be adjusted to reflect the codon usage of thatparticular host, thus leading to higher levels of expression of theRsGluCl channel protein in the host. In other words, this redundancy inthe various codons which code for specific amino acids is within thescope of the present invention. Therefore, this invention is alsodirected to those DNA sequences which encode RNA comprising alternativecodons which code for the eventual translation of the identical aminoacid, as shown below:

[0079] A=Ala=Alanine: codons GCA, GCC, GCG, GCU

[0080] C=Cys=Cysteine: codons UGC, UGU

[0081] D=Asp=Aspartic acid: codons GAC, GAU

[0082] E=Glu=Glutamic acid: codons GAA, GAG

[0083] F=Phe=Phenylalanine: codons UUC, UUU

[0084] G=Gly=Glycine: codons GGA, GGC, GGG, GGU

[0085] H=His =Histidine: codons CAC, CAU

[0086] I=Ile =Isoleucine: codons AUA, AUC, AUU

[0087] K=Lys=Lysine: codons AAA, AAG

[0088] L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU

[0089] M=Met=Methionine: codon AUG

[0090] N=Asp=Asparagine: codons AAC, AAU

[0091] P=Pro=Proline: codons CCA, CCC, CCG, CCU

[0092] Q=Gln=Glutamine: codons CAA, CAG

[0093] R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

[0094] S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU

[0095] T=Thr=Threonine: codons ACA, ACC, ACG, ACU

[0096] V=Val=Valine: codons GUA, GUC, GUG, GUU

[0097] W=Trp=Tryptophan: codon UGG

[0098] Y=Tyr=Tyrosine: codons UAC, UAU

[0099] Therefore, the present invention discloses codon redundancy whichmay result in differing DNA molecules expressing an identical protein.For purposes of this specification, a sequence bearing one or morereplaced codons will be defined as a degenerate variation. Anothersource of sequence variation may occur through RNA editing, as discussedinfra. Such RNA editing may result in another form of codon redundancy,wherein a change in the open reading frame does not result in an alteredamino acid residue in the expressed protein. Also included within thescope of this invention are mutations either in the DNA sequence or thetranslated protein which do not substantially alter the ultimatephysical properties of the expressed protein. For example, substitutionof valine for leucine, arginine for lysine, or asparagine for glutaminemay not cause a change in functionality of the polypeptide.

[0100] It is known that DNA sequences coding for a peptide may bealtered so as to code for a peptide having properties that are differentthan those of the naturally occurring peptide. Methods of altering theDNA sequences include but are not limited to site directed mutagenesis.Examples of altered properties include but are not limited to changes inthe affinity of an enzyme for a substrate or a receptor for a ligand.

[0101] Included in the present invention are DNA sequences thathybridize to SEQ ID NOs:1, 3, 5 and 7 under stringent conditions. By wayof example, and not limitation, a procedure using conditions of highstringency is as follows: Prehybridization of filters containing DNA iscarried out for 2 hours to overnight at 65° C. in buffer composed of 6×SSC, 5× Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA.Filters are hybridized for 12 to 48 hrs at 65° C. in prehybridizationmixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpmof ³²P-labeled probe. Washing of filters is done at 37° C. for 1 hr in asolution containing 2× SSC, 0.1% SDS. This is followed by a wash in 0.1×SSC, 0.1% SDS at 50° C. for 45 min. before autoradiography. Otherprocedures using conditions of high stringency would include either ahybridization step carried out in 5×SSC, 5× Denhardt's solution, 50%formamide at 42° C. for 12 to 48 hours or a washing step carried out in0.2× SSPE, 0.2% SDS at 65° C. for 30 to 60 minutes. Reagents mentionedin the foregoing procedures for carrying out high stringencyhybridization are well known in the art. Details of the composition ofthese reagents can be found in, e.g., Sambrook et al., 1989, MolecularCloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. In addition to the foregoing, other conditions of highstringency which may be used are well known in the art.

[0102] “Identity” is a measure of the identity of nucleotide sequencesor amino acid sequences. In general, the sequences are aligned so thatthe highest order match is obtained. “Identity” per se has anart-recognized meaning and can be calculated using published techniques.See, e.g.,: (Computational Molecular Biology, Lesk, A. M., ed. OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds.. Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). While there exists a number of methods to measure identitybetween two polynucleotide or polypeptide sequences, the term “identity”is well known to skilled artisans (Carillo and Lipton, 1988, SIAM J.Applied Math 48:1073). Methods commonly employed to determine identityor similarity between two sequences include, but are not limited to,those disclosed in Guide to Huge Computers, Martin J. Bishop, ed.,Academic Press, San Diego, 1994, and Carillo and Lipton, 1988, SIAM J.Applied Math 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, et al, 1984, NucleicAcids Research 12(1):387), BLASTN, and FASTA (Altschul, et al., 1990, J.Mol. Biol. 215:403).

[0103] As an illustration, by a polynucleotide having a nucleotidesequence having at least, for example, 95% “identity” to a referencenucleotide sequence of SEQ ID NO:1 is intended that the nucleotidesequence of the polynucleotide is identical to the reference sequenceexcept that the polynucleotide sequence may include up to five pointmutations or alternative nucleotides per each 100 nucleotides of thereference nucleotide sequence of SEQ ID NO:1. In other words, to obtaina polynucleotide having a nucleotide sequence at least 95% identical toa reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations or alternative nucleotide substitutions of the referencesequence may occur at the 5′ or 3 ′ terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence. One source of such a “mutation” or change which results in aless than 100% identity may occur through RNA editing. The process ofRNA editing results in modification of an mRNA molecule such that use ofthat modified mRNA as a template to generate a cloned cDNA may result inone or more nucleotide changes, which may or may not result in a codonchange. This RNA editing is known to be catalyzed by an RNA editase.Such an RNA editase is RNA adenosine deaminase, which converts anadenosine residue to an inosine residue, which tends to mimic a cytosineresidue. To this end, conversion of an mRNA residue from A to I willresult in A to G transitions in the coding and noncoding regions of acloned cDNA (e.g., see Hanrahan et al, 1999, Annals New York Acad. Sci.868:51-66; for a review see Bass, 1997, TIBS 22: 157-162). Similarly, bya polypeptide having an amino acid sequence having at least, forexample, 95% identity to a reference amino acid sequence of SEQ ID NO:2is intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of SEQ ID NO:2. In other words, to obtain apolypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceof anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. Again, as noted above,RNA editing may result in a codon change which will result in anexpressed protein which differs in “identity” from other proteinsexpressed from “non-RNA edited” transcripts, which correspond directlyto the open reading frame of the genomic sequence. The open readingframe of the T12 and T82 clones are identical, save for a singlenucleotide change which results in a single amino acid change(T12-“gag”/Glu v. T82-“aag”/Lys at amino acid residue 447 of SEQ ID NOs:2 and 4). The T12/T82 clone shows about a 57% identity with the B1 cloneat the nucleotide level whereas the T32 clone shows about a 57% identitywith the B1 clone at the nucleotide level.

[0104] The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification. The nucleic acid molecules of the present inventionencoding a RsGluCl channel protein, in whole or in part, can be linkedwith other DNA molecules, i.e, DNA molecules to which the RsGluCl codingsequence are not naturally linked, to form “recombinant DNA molecules”which encode a respective RsGluCl channel protein. The novel DNAsequences of the present invention can be inserted into vectors whichcomprise nucleic acids encoding RsGluCl or a functional equivalent.These vectors may be comprised of DNA or RNA; for most cloning purposesDNA vectors are preferred. Typical vectors include plasmids, modifiedviruses, bacteriophage, cosmids, yeast artificial chromosomes, and otherforms of episomal or integrated DNA that can encode a RsGluCl channelprotein. It is well within the purview of the skilled artisan todetermine an appropriate vector for a particular gene transfer or otheruse.

[0105] The present invention also relates to a substantially purifiedform of a respective RsGluCl channel protein, which comprise the aminoacid sequence disclosed in FIG. 2, FIG. 4, FIG. 6 and FIG. 8, and as setforth in SEQ ID NOs:2, 4, 6, and 8, respectively. The disclosed RsGluClproteins contain an open reading frame of 450 amino acids (T12 and T82,SEQ ID NOs: 2 and 4, respectively), 517 amino acids (T32, SEQ ID NO: 6)and 418 amino acids (SEQ ID NO:8) in length, as shown in FIGS. 2, 4, 6,and 8, and as follows: T12: MSVHSWRFCV PLVALAFFLL ILLSCPSAWG KANFRAIEKRILDSIIGQGR YDCRIRPMGI (SEQ ID NO:2) NNTDGPALVR VNIFVRSIGR IDDVTMEYTVQMTFREQWRD ERLQYDDLGG QVRYLTLTEP DKLWKPDLFF SNEKEGHFHN IIMPNVLLRIHPNGDVLFSI RISLVLSCPM NLKFYPLDKQ ICSIVMVSYG YTTEDLVFLW KEGDPVQVTKNLHLPRFTLE RFQTDYCTSR TNTGEYSCLR VDLVFKREFS YYLIQIYIPC CMLVIVSMVSFWLDPTSIPA RVSLGVTTLL TMATQISGIN ASLPPVSYTK AIDVWTGVCL TFVFGALLEFALVNYASRSD SRRQNMQKQK QRKWELEPPL DSDHLEDGAT TFAMRPLVHH HGELHADKLRQCEVHMKTPK TNLCKAWLSR FPTRSKRIDV VSRIFFPLMF ALFNLVYWTT YLFREDEEDE*; T82:MSVHSWRFCV PLVALAFFLL ILLSCPSAWG KANFRAIEKR ILDSIIGQGR YDCRIRPMGI (SEQID NO:4) NNTDGPALVR VNIFVRSIGR IDDVTMEYTV QMTFREQWRD ERLQYDDLGGQVRYLTLTEP DKLWKPDLFF SNEKEGHFHN IIMPNVLLRI HPNGDVLFSI RISLVLSCPMNLKFYPLDKQ ICSIVMVSYG YTTEDLVFLW KEGDPVQVTK NLHLPRFTLE RFQTDYCTSRTNTGEYSCLR VDLVFKREFS YYLIQIYIPC CMLVIVSWVS FWLDPTSIPA RVSLGVTTLLTMATQISGIN ASLPPVSYTK AIDVWTGVCL TFVFGALLEF ALVNYASRSD SRRQNMQKQKQRKWELEPPL DSDHLEDGAT TFAMRPLVHH HGELHADKLR QCEVHMKTPK TNLCKAWLSRFPTRSKRIDV VSRIFFPLMF ALFNLVYWTT YLFREDKEDE*; T32: MSVHSWRFCV PLVALAFFLLILLSCPSAWA ETLPTPPTRG QGGVPVAAAM LLGKQQSSRY (SEQ ID NO:6) QDKEGKANFRAIEKRILDSI IGQGRYDCRI RPMGINNTDG PALVRVNIFV RSIGRIDDVT MEYTVQMTFREQWRDERLQY DDLGGQVRYL TLTEPDKLWK PDLFFSNEKE GHFHNIIMPN VLLRIHPNGDVLFSIRISLV LSCPMNLKFY PLDKQICSIV MVSYGYTTED LVFLWKEGDP VQVTKNLHLPRFTLERFQTD YCTSRTNTGE YSCLRVDLVF KREFSYYLIQ IYIPCCMLVI VSWVSFWLDPTSIPARVSLG VTTLLTMATQ ISGINASLPP VSYTKAIDVW TGVCLTFVFG ALLEFALVNYASRSDSRRQN MQKQKQRKWE LEPPLDSDHL EDGATTFAMV SSGEPAGLMA RTWPPPPLPPNMAAGSAQAG ARPLVHHHGE LHADKLRQCE VHMKTPKTNL CKAWLSRFPT RSKRIDVVSRIFFPLVFALF NLVYWTTYLF REDEEDE*; and, B1: MFGVPCSRAC RLVVVIAAFCWPPALPLVPG GVSSRANDLD ILDELLKNYD RRALPSSHLG (SEQ ID NO:8) NATIVSCEIYIRSFGSINPS NMDYEVDLYF RQSWLDERLR KSTLSRPLDL NDPKLVQMIW KPEVFFANAKHAEFQYVTVP NVLVRINPTG IILYMLRLKL RFSCMMDLYR YPMDSQVCSI EIASFSKTTEELLLKWSESQ PVVLFDNLKL PQFEIEKVNT SLCKEKFHIG EYSCLKADFY LQRSLGYHMVQTYLPTTLIV VISWVSFWLD YLWRRLPNKR PSSDVPVTDI PSDGSKHDIA AQLVLDKNGHTEVRTLVQAM PRSVGKVKAK QIDQLSRVAF PALFLLFNLV YWPYYIKS.

[0106] The open reading frames of the T12 and T82 clones are identical,save for a single nucleotide change which results in a single amino acidchange at residue 447 of SEQ ID NOs: 2 and 4. The T32 open reading framecontains two addition exons when compared to the T12/T82 reading frame,which result in a 35 amino acid insertion in the amino terminal regionof the T32 protein (amino acid residue 30-64 of SEQ ID NO:6) and another32 amino acid insertion within the COOH-terminal region (amino acidresidue 410-441). The T12/T82 clones show about a 57% identity with theB1 clone at the nucleotide level whereas the T32 clone shows about a 57%identity with the B1 clone at the nucleotide level.

[0107] The present invention also relates to biologically activefragments and/or mutants of the RsGluCl1 and RsGluCl2 proteinscomprising the amino acid sequence as set forth in SEQ ID NOs:2, 4, 6,and 8, including but not necessarily limited to amino acidsubstitutions, deletions, additions, amino terminal truncations andcarboxy-terminal truncations such that these mutations provide forproteins or protein fragments of diagnostic, therapeutic or prophylacticuse and would be useful for screening for agonists and/or antagonists ofRsGluCl function.

[0108] To this end, a preferred aspect of the present invention is afunctional RsGluCl channel receptor, comprised of either a singlechannel protein or a channel comprising multiple subunits, referred toherein as a homomultimeric channel or a heteromultimeric channel.Therefore, a single channel may be comprised of a protein as disclosedin SEQ ID NOs: 2, 4, 6 or 8, or a biologically active equivalent thereof(i.e., an altered channel protein which still functions in a similarfashion to that of a wild-type channel receptor). A homomultimericchannel receptor complex will comprise more than one polypeptideselected from the disclosed group of SEQ ID NOs: 2, 4, 6 and 8, as wellas biologically active equivalents. A heteromultimeric channel receptorcomplex will comprise multiple subunits wherein at least 2 of the 3proteins disclosed herein contribute to channel formation, or where atleast one of the proteins associates with additional proteins or channelcomponents to provide for an active channel receptor complex. Therefore,the present invention additionally relates to substantially purifiedchannels as described herein, as well as substantially purified membranepreparations, partially purified membrane preparations, or cell lysateswhich contain the functional single, homomultimeric or heteromultimericchannels described herein. These substantially purified, fully processedGluCl channel proteins may be obtained from a recombinant host cellcontaining a DNA expression vector comprises a nucleotide sequence asset forth in SEQ ID NOs: 1, 3, 5, and/or 7, and expresses the respectiveRsGluCl precursor protein. It is especially preferred is that therecombinant host cell be a eukaryotic host cell, including but notlimited to a mammalian cell line, an insect cell line such as an S2 cellline, or Xenopus oocytes, as noted above.

[0109] As with many proteins, it is possible to modify many of the aminoacids of RsGluCl channel protein and still retain substantially the samebiological activity as the wild type protein. Thus this inventionincludes modified RsGluCl polypeptides which have amino acid deletions,additions, or substitutions but that still retain substantially the samebiological activity as a respective, corresponding RsGluCl. It isgenerally accepted that single amino acid substitutions do not usuallyalter the biological activity of a protein (see, e.g., Molecular Biologyof the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/CummingsPublishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science244:1081-1085). Accordingly, the present invention includes polypeptideswhere one amino acid substitution has been made in SEQ ID NO:2, 4, 6,and/or 8, wherein the polypeptides still retain substantially the samebiological activity as a corresponding RsGluCl protein. The presentinvention also includes polypeptides where two or more amino acidsubstitutions have been made in SEQ ID NO:2, 4, 6, or 8, wherein thepolypeptides still retain substantially the same biological activity asa corresponding RsGluCl protein. In particular, the present inventionincludes embodiments where the above-described substitutions areconservative substitutions.

[0110] One skilled in the art would also recognize that polypeptidesthat are functional equivalents of RsGluCl and have changes from theRsGluCl amino acid sequence that are small deletions or insertions ofamino acids could also be produced by following the same guidelines,(i.e, minimizing the differences in amino acid sequence between RsGluCland related proteins. Small deletions or insertions are generally in therange of about 1 to 5 amino acids. The effect of such small deletions orinsertions on the biological activity of the modified RsGluClpolypeptide can easily be assayed by producing the polypeptidesynthetically or by making the required changes in DNA encoding RsGluCland then expressing the DNA recombinantly and assaying the proteinproduced by such recombinant expression.

[0111] The present invention also includes truncated forms of RsGluClwhich contain the region comprising the active site of the enzyme. Suchtruncated proteins are useful in various assays described herein, forcrystallization studies, and for structure-activity-relationshipstudies.

[0112] The present invention also relates to membrane-containing crudelysates or substantially purified subcellular membrane fractions fromthe recombinant host cells (both prokaryotic and eukaryotic as well asboth stably and transiently transformed or transfected cells) whichcontain the nucleic acid molecules of the present invention. Theserecombinant host cells express RsGluCl or a functional equivalent, whichbecomes post translationally associated with the cell membrane in abiologically active fashion. These subcellular membrane fractions willcomprise either wild-type or mutant forms of RsGluCl at levelssubstantially above endogenous levels and hence will be useful in assaysto select modulators of RsGluCl proteins or channels. In other words, aspecific use for such subcellular membranes involves expression ofRsGluCl within the recombinant cell followed by isolation andsubstantial purification of the membranes away from other cellularcomponents and subsequent use in assays to select for modulators, suchas agonist or antagonists of the protein or biologically active channelcomprising one or more of the proteins disclosed herein. Alternatively,the lysed cells, containing the membranes, may be used directly inassays to select for modulators of the recombinantly expressedprotein(s) disclosed herein. Therefore, another preferred aspect of thepresent invention relates to a substantially purified membranepreparation or lysed recombinant cell components which includemembranes, which has been obtained from a recombinant host celltransformed or transfected with a DNA expression vector which comprisesand appropriately expresses a complete open reading frame as set forthin SEQ ID NOs: 1, 3, 5, and/or 7, resulting in a functional form of therespective RsGluCl channel. It is especially preferred is that therecombinant host cell be a eukaryotic host cell, including but notlimited to a mammalian cell line, an insect cell line such as an S2 cellline.

[0113] The present invention also relates to isolated nucleic acidmolecules which are fusion constructions expressing fusion proteinsuseful in assays to identify compounds which modulate wild-type RsGluClactivity, as well as generating antibodies against RsGluCl. One aspectof this portion of the invention includes, but is not limited to,glutathione S-transferase (GST)-RsGluCl fusion constructs. RecombinantGST-RsGluCl fusion proteins may be expressed in various expressionsystems, including Spodoptera frugiperda (Sf21) insect cells(Invitrogen) using a baculovirus expression vector (pAcG2T, Pharmingen).Another aspect involves RsGluCl fusion constructs linked to variousmarkers, including but not limited to GFP (Green fluorescent protein),the MYC epitope, and GST. Again, any such fusion constructs may beexpressed in the cell line of interest and used to screen for modulatorsof one or more of the RsGluCl proteins disclosed herein.

[0114] A preferred aspect for screening for modulators of RsGluClchannel activity is an expression system for theelectrophysiological-based assays for measuring glutamate-gated chloridechannel activity comprising injecting the DNA molecules of the presentinvention into Xenopus laevis oocytes. The general use of Xenopusoocytes in the study of ion channel activity is known in the art(Dascal, 1987, Crit. Rev. Biochem. 22: 317-317; Lester, 1988, Science241: 1057-1063; see also Methods of Enzymology, Vol. 207, 1992, Ch.14-25, Rudy and Iverson, ed., Academic Press, Inc., New York). Animproved method exists for measuring channel activity and modulation byagonists and/or antagonists which is several-fold more sensitive thanprevious techniques. The Xenopus oocytes are injected with nucleic acidmaterial, including but not limited to DNA, mRNA or cRNA which encode agated-channel, wherein channel activity may be measured as well asresponse of the channel to various modulators. Ion channel activity ismeasured by utilizing a holding potential more positive than thereversal potential for chloride (i.e, greater than −30 mV), preferablyabout 0 mV. This alteration in assay measurement conditions results in a10-fold increase in sensitivity of the assay to modulation by ivermectinphosphate. Therefore, this improved assay allows screening and selectingfor compounds which modulate GluCl activity at levels which werepreviously thought to be undetectable.

[0115] Any of a variety of procedures may be used to clone RsGluCl.These methods include, but are not limited to, (1) a RACE PCR cloningtechnique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998-9002). 5′ and/or 3′ RACE may be performed to generate a full-lengthcDNA sequence. This strategy involves using gene-specificoligonucleotide primers for PCR amplification of RsGluCl cDNA. Thesegene-specific primers are designed through identification of anexpressed sequence tag (EST) nucleotide sequence which has beenidentified by searching any number of publicly available nucleic acidand protein databases; (2) direct functional expression of the RsGluClcDNA following the construction of a RsGluCl-containing cDNA library inan appropriate expression vector system; (3) screening aRsGluCl-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a labeled degenerate oligonucleotide probedesigned from the amino acid sequence of the RsGluCl protein; (4)screening a RsGluCl-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA encoding theRsGluCl protein. This partial cDNA is obtained by the specific PCRamplification of RsGluCl DNA fragments through the design of degenerateoligonucleotide primers from the amino acid sequence known for otherGluCl channels which are related to the RsGluCl protein; (5) screening aRsGluCl-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a partial cDNA or oligonucleotide withhomology to a RsGluCl protein. This strategy may also involve usinggene-specific oligonucleotide primers for PCR amplification of RsGluClcDNA identified as an EST as described above; or (6) designing 5′ and 3′gene specific oligonucleotides using SEQ ID NO: 1, 3, and 5 as atemplate so that either the full-length cDNA may be generated by knownRACE techniques, or a portion of the coding region may be generated bythese same known RACE techniques to generate and isolate a portion ofthe coding region to use as a probe to screen one of numerous types ofcDNA and/or genomic libraries in order to isolate a full-length versionof the nucleotide sequence encoding RsGluCl. Alternatively, the RsGluCl1and RsGluCl2 cDNAs of the present invention may be cloned as describedin Example Section 1. For RsGluCl1 cDNA clones, adult brown dog tickpolyA⁺ RNA was isolated using the Poly(A)Pure™ mRNA Isolation Kit(Ambion). Tick cDNA was synthesized using oligo-dT primers and the ZAPcDNA® Synthesis Kit (Stratagene),and cDNA>1 kb was selected using cDNASize Fractionation Columns (BRL). A tick cDNA library was constructed inthe Lambda ZAP® II vector using the GIGAPACK® III Gold Cloning Kit(Stratagene). A. Drosophila GluCl cDNA fragment spanning the M1 to M3region was used in a low-stringency screen of the tick cDNA library.Filters were exposed for eleven days and six positives were isolated forsequence analysis. Three of the clones (T12, T82 and T32) encodeGluCl-related proteins and were sequenced on both ends. For isolation ofthe RsGluCl2 cDNAs, most molecular procedures were again performedfollowing standard procedures available in references such as Ausubelet. al. (1992. Short protocols in molecular biology. F. M. Ausubel etal.,—2^(nd). ed. (John Wiley & Sons), and Sambrook et al. (1989.Molecular cloning. A laboratory manual. J. Sambrook, E. F. Fritsch, andT. Maniatis—2^(nd) ed. (Cold Spring Harbor Laboratory Press). Poly (A)+RNA was isolated from Tick heads. First strand cDNA was synthesized from50 ng RNA using a SUPERSCRIPT preamplification System (LifeTechnologies). A tenth of the first strand reaction was used for PCR.The degenerate oligos utilized were designed based on sequences obtainedfrom C. elegans, Drosophila, and Flea (C. felis) GluCls: Two PCR rounds,using the combinations “27F2+3AF1, then 27F2+3BF2” were performed. Onetenth of the PCR reaction products was tested by Southern blot analysis,in order to identify and prevent the PCR-cloning of contaminatingsequences. Novel PCR products of the appropriate size were cloned intothe pCR2.1 plasmid vector using a “TA” cloning kit (Invitrogen, Inc.).Following sequence analysis (ABI Prism, PE Applied Biosystems), selectedPCR clone inserts were radiolabelled and used as probes to screen a cDNAlibrary generated into the Uni-ZAP® vector (Stratagene, Inc.) from usingthe RNA preparation mentioned above. Sequences from full-length cDNAclones were analysed using the GCG Inc. package. Subcloning of RsGluCl2into a mammalian expression vector was done by excision of an 1.85 kbcoding-region-containing fragment (XhoI-EcoRI digest) from the originalinsert of clone RsGluCl2 B1 from the UniZap® (pBS plasmid, followed byligation into the TetSplice® vector (Life Technologies Inc.).

[0116] It is readily apparent to those skilled in the art that othertypes of libraries, as well as libraries constructed from other celltypes—or species types, may be useful for isolating a RsGluCl-encodingDNA or a RsGluCl homologue. Other types of libraries include, but arenot limited to, cDNA libraries derived from other brown dog tick celltypes.

[0117] It is readily apparent to those skilled in the art that suitablecDNA libraries may be prepared from cells or cell lines which haveRsGluCl activity. The selection of cells or cell lines for use inpreparing a cDNA library to isolate a cDNA encoding RsGluCl may be doneby first measuring cell-associated RsGluCl activity using any knownassay available for such a purpose.

[0118] Preparation of cDNA libraries can be performed by standardtechniques well known in the art. Well known cDNA library constructiontechniques can be found for example, in Sambrook et al., 1989, MolecularCloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. Complementary DNA libraries may also be obtained fromnumerous commercial sources, including but not limited to ClontechLaboratories, Inc. and Stratagene.

[0119] It is also readily apparent to those skilled in the art that DNAencoding RsGluCl may also be isolated from a suitable genomic DNAlibrary. Construction of genomic DNA libraries can be performed bystandard techniques well known in the art. Well known genomic DNAlibrary construction techniques can be found in Sambrook, et al., supra.One may prepare genomic libraries, especially in P1 artificialchromosome vectors, from which genomic clones containing the RsGluCl canbe isolated, using probes based upon the RsGluCl nucleotide sequencesdisclosed herein. Methods of preparing such libraries are known in theart (Ioannou et al., 1994, Nature Genet. 6:84-89).

[0120] In order to clone a RsGluCl gene by one of the preferred methods,the amino acid sequence or DNA sequence of a RsGluCl or a homologousprotein may be necessary. To accomplish this, a respective RsGluClchannel protein may be purified and the partial amino acid sequencedetermined by automated sequenators. It is not necessary to determinethe entire amino acid sequence, but the linear sequence of two regionsof 6 to 8 amino acids can be determined for the PCR amplification of apartial RsGluCl DNA fragment. Once suitable amino acid sequences havebeen identified, the DNA sequences capable of encoding them aresynthesized. Because the genetic code is degenerate, more than one codonmay be used to encode a particular amino acid, and therefore, the aminoacid sequence can be encoded by any of a set of similar DNAoligonucleotides. Only one member of the set will be identical to theRsGluCl sequence but others in the set will be capable of hybridizing toRsGluCl DNA even in the presence of DNA oligonucleotides withmismatches. The mismatched DNA oligonucleotides may still sufficientlyhybridize to the RsGluCl DNA to permit identification and isolation ofRsGluCl encoding DNA. Alternatively, the nucleotide sequence of a regionof an expressed sequence may be identified by searching one or moreavailable genomic databases. Gene-specific primers may be used toperform PCR amplification of a cDNA of interest from either a cDNAlibrary or a population of cDNAs. As noted above, the appropriatenucleotide sequence for use in a PCR-based method may be obtained fromSEQ ID NO: 1, 3, 5, or 7 either for the purpose of isolating overlapping5′ and 3′ RACE products for generation of a full-length sequence codingfor RsGluCl, or to isolate a portion of the nucleotide sequence codingfor RsGluCl for use as a probe to screen one or more cDNA- orgenomic-based libraries to isolate a full-length sequence encodingRsGluCl or RsGluCl-like proteins.

[0121] This invention also includes vectors containing a RsGluCl gene,host cells containing the vectors, and methods of making substantiallypure RsGluCl protein comprising the steps of introducing the RsGluClgene into a host cell, and cultivating the host cell under appropriateconditions such that RsGluCl is produced. The RsGluCl so produced may beharvested from the host cells in conventional ways. Therefore, thepresent invention also relates to methods of expressing the RsGluClprotein and biological equivalents disclosed herein, assays employingthese gene products, recombinant host cells which comprise DNAconstructs which express these proteins, and compounds identifiedthrough these assays which act as agonists or antagonists of RsGluClactivity.

[0122] The cloned RsGluCl cDNA obtained through the methods describedabove may be recombinantly expressed by molecular cloning into anexpression vector (such as pcDNA3.neo, pcDNA3.1, pCR2.1, pBlueBacHis2 orpLITMUS28, as well as other examples, listed infra) containing asuitable promoter and other appropriate transcription regulatoryelements, and transferred into prokaryotic or eukaryotic host cells toproduce recombinant RsGluCl. Expression vectors are defined herein asDNA sequences that are required for the transcription of cloned DNA andthe translation of their mRNAs in an appropriate host. Such vectors canbe used to express eukaryotic DNA in a variety of hosts such asbacteria, blue green algae, plant cells, insect cells and animal cells.Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector should contain: an origin of replicationfor autonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one which causes mRNAs to be initiated at highfrequency. To determine the RsGluCl cDNA sequence(s) that yields optimallevels of RsGluCl, cDNA molecules including but not limited to thefollowing can be constructed: a cDNA fragment containing the full-lengthopen reading frame for RsGluCl as well as various constructs containingportions of the cDNA encoding only specific domains of the protein orrearranged domains of the protein. All constructs can be designed tocontain none, all or portions of the 5′ and/or 3′ untranslated region ofa RsGluCl cDNA. The expression levels and activity of RsGluCl can bedetermined following the introduction, both singly and in combination,of these constructs into appropriate host cells. Following determinationof the RsGluCl cDNA cassette yielding optimal expression in transientassays, this RsGluCl cDNA construct is transferred to a variety ofexpression vectors (including recombinant viruses), including but notlimited to those for mammalian cells, plant cells, insect cells,oocytes, bacteria, and yeast cells. Techniques for such manipulationscan be found described in Sambrook, et al., supra, are well known andavailable to the artisan of ordinary skill in the art. Therefore,another aspect of the present invention includes host cells that havebeen engineered to contain and/or express DNA sequences encoding theRsGluCl. An expression vector containing DNA encoding a RsGluCl-likeprotein may be used for expression of RsGluCl in a recombinant hostcell. Such recombinant host cells can be cultured under suitableconditions to produce RsGluCl or a biologically equivalent form.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.Commercially available mammalian expression vectors which may besuitable for recombinant RsGluCl expression, include but are not limitedto, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega),pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs),pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMC1neo(Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146),pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565). Also, a variety ofbacterial expression vectors may be used to express recombinant RsGluClin bacterial cells. Commercially available bacterial expression vectorswhich may be suitable for recombinant RsGluCl expression include, butare not limited to pCR2.1 (Invitrogen), pET11a (Novagen), lambda gt11(Invitrogen), and pKK223-3 (Pharmacia). In addition, a variety of fungalcell expression vectors may be used to express recombinant RsGluCl infungal cells. Commercially available fungal cell expression vectorswhich may be suitable for recombinant RsGluCl expression include but arenot limited to pYES2 (Invitrogen) and Pichia expression vector(Invitrogen). Also, a variety of insect cell expression vectors may beused to express recombinant protein in insect cells. Commerciallyavailable insect cell expression vectors which may be suitable forrecombinant expression of RsGluCl include but are not limited topBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).

[0123] Recombinant host cells may be prokaryotic or eukaryotic,including but not limited to, bacteria such as E. coli, fungal cellssuch as yeast, mammalian cells including, but not limited to, cell linesof bovine, porcine, monkey and rodent origin; and insect cells includingbut not limited to R. sanguineus and silkworm derived cell lines. Forinstance, one insect expression system utilizes Spodoptera frugiperda(Sf21) insect cells (Invitrogen) in tandem with a baculovirus expressionvector (pAcG2T, Pharmingen). Also, mammalian species which may besuitable and which are commercially available, include but are notlimited to, L cells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2),Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1(ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCCCCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL171) and CPAE (ATCC CCL 209).

[0124] The specificity of binding of compounds showing affinity forRsGluCl is shown by measuring the affinity of the compounds forrecombinant cells expressing the cloned receptor or for membranes fromthese cells, which form a functional single, homomultimeric orheteromultimeric membrane channel. Expression of the cloned receptor andscreening for compounds that bind to RsGluCl or that inhibit the bindingof a known, radiolabeled ligand of RsGluCl to these cells, or membranesprepared from these cells, provides an effective method for the rapidselection of compounds with high affinity for RsGluCl. Such ligands neednot necessarily be radiolabeled but can also be nonisotopic compoundsthat can be used to displace bound radiolabeled compounds or that can beused as activators in functional assays. Compounds identified by theabove method are likely to be agonists or antagonists of RsGluCl and maybe peptides, proteins, or non-proteinaceous organic or inorganicmolecules.

[0125] A preferred aspect for screening for modulators of RsGluClchannel activity is an expression system forelectrophysiologically-based assays for measuring ligand gated channelactivity (such as GluCl channel activity) comprising injecting the DNAor RNA molecules of the present invention into Xenopus laevis oocytes.The general use of Xenopus oocytes in the study of ion channel activityis known in the art (Dascal, 1987, Crit. Rev. Biochem. 22: 317-317;Lester, 1988, Science 241: 1057-1063; see also Methods of Enzymology,Vol. 207, 1992, Ch. 14-25, Rudy and Iverson, ed., Academic Press, Inc.,New York). The Xenopus oocytes are injected with nucleic acid material,including but not limited to DNA, mRNA or cRNA which encode a ligandgated-channel, whereafter channel activity may be measured as well asresponse of the channel to various modulators.

[0126] Accordingly, the present invention is directed to methods forscreening for compounds which modulate the expression of DNA or RNAencoding a RsGluCl protein as well as compounds which effect thefunction of the RsGluCl protein. Methods for identifying agonists andantagonists of other receptors are well known in the art and can beadapted to identify agonists and antagonists of a RsGluCl channel. Forexample, Cascieri et al. (1992, Molec. Pharmacol. 41:1096-1099) describea method for identifying substances that inhibit agonist binding to ratneurokinin receptors and thus are potential agonists or antagonists ofneurokinin receptors. The method involves transfecting COS cells withexpression vectors containing rat neurokinin receptors, allowing thetransfected cells to grow for a time sufficient to allow the neurokininreceptors to be expressed, harvesting the transfected cells andresuspending the cells in assay buffer containing a known radioactivelylabeled agonist of the neurokinin receptors either in the presence orthe absence of the substance, and then measuring the binding of theradioactively labeled known agonist of the neurokinin receptor to theneurokinin receptor. If the amount of binding of the known agonist isless in the presence of the substance than in the absence of thesubstance, then the substance is a potential ligand of the neurokininreceptor. Where binding of the substance such as an agonist orantagonist to RsGluCl is measured, such binding can be measured byemploying a labeled ligand. The ligand can be labeled in any convenientmanner known to the art, e.g., radioactively, fluorescently,enzymatically.

[0127] Therefore, the present invention is directed to methods forscreening for compounds which modulate the expression of DNA or RNAencoding a RsGluCl protein. Compounds which modulate these activitiesmay be DNA, RNA, peptides, proteins, or non-proteinaceous organic orinorganic molecules. Compounds may modulate by increasing or attenuatingthe expression of DNA or RNA encoding RsGluCl, or the function of theRsGluCl-based channels. Compounds that modulate the expression of DNA orRNA encoding RsGluCl or the biological function thereof may be detectedby a variety of assays. The assay may be a simple “yes/no” assay todetermine whether there is a change in expression or function. The assaymay be made quantitative by comparing the expression or function of atest sample with the levels of expression or function in a standardsample. Kits containing RsGluCl, antibodies to RsGluCl, or modifiedRsGluCl may be prepared by known methods for such uses.

[0128] To this end, the present invention relates in part to methods ofidentifying a substance which modulates RsGluCl receptor activity, whichinvolves:

[0129] (a) adding a test substance in the presence and absence of aRsGluCl receptor protein wherein said RsGluCl receptor protein comprisesthe amino acid sequence as set forth in SEQ ID NOs: 2, 6 and/or 8; and,

[0130] (b) measuring and comparing the effect of the test substance inthe presence and absence of the RsGluCl receptor protein or respectivefunctional channel.

[0131] In addition, several specific embodiments are disclosed herein toshow the diverse types of screening or selection assays which theskilled artisan may utilize in tandem with an expression vectordirecting the expression of the RsGluCl receptor protein. Methods foridentifying ligands of other receptors are well known in the art and canbe adapted to ligands of RsGluCl. Therefore, these embodiments arepresented as examples and not as limitations. To this end, the presentinvention includes assays by which RsGluCl modulators (such as agonistsand antagonists) may be identified. Accordingly, the present inventionincludes a method for determining whether a substance is a potentialagonist or antagonist of RsGluCl that comprises:

[0132] (a) transfecting or transforming cells with an expression vectorthat directs expression of RsGluCl in the cells, resulting in testcells;

[0133] (b) allowing the test cells to grow for a time sufficient toallow RsGluCl to be expressed and for a functional channel to begenerated;

[0134] (c) exposing the cells to a labeled ligand of RsGluCl in thepresence and in the absence of the substance;

[0135] (d) measuring the binding of the labeled ligand to the RsGluClchannel; where if the amount of binding of the labeled ligand is less inthe presence of the substance than in the absence of the substance, thenthe substance is a potential ligand of RsGluCl.

[0136] The conditions under which step (c) of the method is practicedare conditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Thetest cells may be harvested and resuspended in the presence of thesubstance and the labeled ligand. In a modification of theabove-described method, step (c) is modified in that the cells are notharvested and resuspended but rather the radioactively labeled knownagonist and the substance are contacted with the cells while the cellsare attached to a substratum, e.g., tissue culture plates.

[0137] The present invention also includes a method for determiningwhether a substance is capable of binding to RsGluCl, i.e., whether thesubstance is a potential modulator of RsGluCl channel activation, wherethe method comprises:

[0138] (a) transfecting or transforming cells with an expression vectorthat directs the expression of RsGluCl in the cells, resulting in testcells;

[0139] (b) exposing the test cells to the substance;

[0140] (c) measuring the amount of binding of the substance to RsGluCl;

[0141] (d) comparing the amount of binding of the substance to RsGluClin the test cells with the amount of binding of the substance to controlcells that have not been transfected with RsGluCl;

[0142] wherein if the amount of binding of the substance is greater inthe test cells as compared to the control cells, the substance iscapable of binding to RsGluCl.

[0143] Determining whether the substance is actually an agonist orantagonist can then be accomplished by the use of functional assays,such as an electrophysiological assay described herein.

[0144] The conditions under which step (b) of the method is practicedare conditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Thetest cells are harvested and resuspended in the presence of thesubstance.

[0145] The above described assays may be functional assays, whereelectrophysiological assays (e.g., see Example 2) may be carried out intransfected mammalian cell lines, an insect cell line, or Xenopusoocytes to measure the various effects test compounds may have on theability of a known ligand (such as glutamate) to activate the channel,or for a test compound to modulate activity in and of itself (similar tothe effect of ivermectin on known GluCl channels). Therefore, theskilled artisan will be comfortable adapting the cDNA clones of thepresent invention to known methodology for both initial and secondaryscreens to select for compounds that bind and/or activate the functionalRsGluCl channels of the present invention.

[0146] A preferred method of identifying a modulator of a RsGluClchannel protein comprise firstly contacting a test compound with a R.sanguineus RsGluCl channel protein selected from the group consisting ofSEQ ID NOs:2, 4, 6 and 8; and, secondly measuring the effect of the testcompound on the RsGluCl channel protein. A preferred aspect involvesusing a R. sanguineus RsGluCl protein which is a product of a DNAexpression vector contained within a recombinant host cell.

[0147] Another preferred method of identifying a compound that modulatesRsGluCl glutamate-gated channel protein activity comprises firstlyinjecting into a host cell a population of nucleic acid molecules, atleast a portion of which encodes a R. sanguineus GluCl channel proteinselected from the group consisting of SEQ ID NOs:2, 4, 6 and 8, suchthat expression of said portion of nucleic acid molecules results in anactive ligand-gated channel, secondly measuring host cell membranecurrent in the presence and absense of a test compound. Numeroustemplates may be used, including but not limited to complementary DNA,poly A⁺ messenger RNA and complementary RNA.

[0148] The DNA molecules, RNA molecules, recombinant protein andantibodies of the present invention may be used to screen and measurelevels of RsGluCl. The recombinant proteins, DNA molecules, RNAmolecules and antibodies lend themselves to the formulation of kitssuitable for the detection and typing of RsGluCl. Such a kit wouldcomprise a compartmentalized carrier suitable to hold in closeconfinement at least one container. The carrier would further comprisereagents such as recombinant RsGluCl or anti-RsGluCl antibodies suitablefor detecting RsGluCl. The carrier may also contain a means fordetection such as labeled antigen or enzyme substrates or the like.

[0149] The assays described herein can be carried out with cells thathave been transiently or stably transfected with RsGluCl. The expressionvector may be introduced into host cells via any one of a number oftechniques including but not limited to transformation, transfection,protoplast fusion, and electroporation. Transfection is meant to includeany method known in the art for introducing RsGluCl into the test cells.For example, transfection includes calcium phosphate or calcium chloridemediated transfection, lipofection, infection with a retroviralconstruct containing RsGluCl, and electroporation. The expressionvector-containing cells are individually analyzed to determine whetherthey produce RsGluCl protein. Identification of RsGluCl expressing cellsmay be done by several means, including but not limited to immunologicalreactivity with anti-RsGluCl antibodies, labeled ligand binding, or thepresence of functional, non-endogenous RsGluCl activity.

[0150] The specificity of binding of compounds showing affinity forRsGluCl is shown by measuring the affinity of the compounds forrecombinant cells expressing the cloned receptor or for membranes fromthese cells. Expression of the cloned receptor and screening forcompounds that bind to RsGluCl or that inhibit the binding of a known,ligand of RsGluCl to these cells, or membranes prepared from thesecells, provides an effective method for the rapid selection of compoundswith high affinity for RsGluCl. Such ligands need not necessarily beradiolabeled but can also be nonisotopic compounds that can be used todisplace bound radioactively, fluorescently or enzymatically labeledcompounds or that can be used as activators in functional assays.Compounds identified by the above method are likely to be agonists orantagonists of RsGluCl.

[0151] Therefore, the specificity of binding of compounds havingaffinity for RsGluCl is shown by measuring the affinity of the compoundsfor recombinant cells expressing the cloned receptor or for membranesfrom these cells. Expression of the cloned receptor and screening forcompounds that bind to RsGluCl or that inhibit the binding of a known,radiolabeled ligand of RsGluCl (such as glutamate, ivermectin ornodulisporic acid) to these cells, or membranes prepared from thesecells, provides an effective method for the rapid selection of compoundswith high affinity for RsGluCl. Such ligands need not necessarily beradiolabeled but can also be nonisotopic compounds that can be used todisplace bound radioactively, fluorescently or enzymatically labeledcompounds or that can be used as activators in functional assays.Compounds identified by the above method again are likely to be agonistsor antagonists of RsGluCl. As noted elsewhere in this specification,compounds may modulate by increasing or attenuating the expression ofDNA or RNA encoding RsGluCl, or by acting as an agonist or antagonist ofthe RsGluCl receptor protein. Again, these compounds that modulate theexpression of DNA or RNA encoding RsGluCl or the biological functionthereof may be detected by a variety of assays. The assay may be asimple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function of a test sample with the levels ofexpression or function in a standard sample.

[0152] RsGluCl 1 and/or 2 gated chloride channel functional assaysmeasure one or more ligand-gated chloride channel activities where thechannel is made up in whole, or in part, by the RsGluCl channel. RsGluClchannel activity can be measured using the channel described herein byitself; or as a subunit in combination with one or more additionalligand-gated chloride channel subunits (preferably one or more RsGluCl),where the subunits combine together to provide functional channelactivity. Assays measuring RsGluCl-gated chloride channel activityinclude functional screening using ³⁶Cl, functional screening usingpatch clamp electrophysiology and functional screening using fluorescentdyes. Techniques for carrying out such assays in general are well knownin the art. (See, for example, Smith et al., 1998, European Journal ofPharmacology 159:261-269; Gonzalez and Tsien, 1997, Chemistry & Biology4:269-277; Millar et al., 1994, Proc. R. Soc. Lond. B. 258:307-314; Rauhet al., 1990 TiPS 11:325-329, and Tsien et al., U.S. Pat. No.5,661,035.) Functional assays can be performed using individualcompounds or preparations containing different compounds. A preparationcontaining different compounds where one or more compounds affectRsGluCl channel activity can be divided into smaller groups of compoundsto identify the compound(s) affecting RsGluCl channel activity. In an.embodiment of the present invention a test preparation containing atleast 10 compounds is used in a functional assay. Recombinantly producedRsGluCl channels present in different environments can be used in afunctional assay. Suitable environments include live cells and purifiedcell extracts containing the RsGluCl channel and an appropriate membranefor activity; and the use of a purified RsGluCl channel produced byrecombinant means that is introduced into a different environmentsuitable for measuring RsGluCl channel activity. RsGluCl derivatives canbe used to assay for compounds active at the channel and to obtaininformation concerning different regions of the channel. For example,RsGluCl channel derivatives can be produced where amino acid regions inthe native channel are altered and the effect of the alteration onchannel activity can be measured to obtain information regardingdifferent channel regions.

[0153] Expression of RsGluCl DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes, with microinjection into frog oocytes being preferred.

[0154] Following expression of RsGluCl in a host cell, RsGluCl proteinmay be recovered to provide RsGluCl protein in active form. SeveralRsGluCl protein purification procedures are available and suitable foruse. Recombinant RsGluCl protein may be purified from cell lysates andextracts by various combinations of, or individual application of saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography. In addition, recombinant RsGluClprotein can be separated from other cellular proteins by use of animmunoaffinity column made with monoclonal or polyclonal antibodiesspecific for full-length RsGluCl protein, or polypeptide fragments ofRsGluCl protein.

[0155] Expression of RsGluCl DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes, with microinjection into frog oocytes being preferred.

[0156] Following expression of RsGluCl in a host cell, RsGluCl proteinmay be recovered to provide RsGluCl protein in active form. SeveralRsGluCl protein purification procedures are available and suitable foruse. Recombinant RsGluCl protein may be purified from cell lysates andextracts by various combinations of, or individual application of saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography. In addition, recombinant RsGluClprotein can be separated from other cellular proteins by use of animmunoaffinity column made with monoclonal or polyclonal antibodiesspecific for full-length RsGluCl protein, or polypeptide fragments ofRsGluCl protein.

[0157] Polyclonal or monoclonal antibodies may be raised againstRsGluCl1 or RsGluCl2 or a synthetic peptide (usually from about 9 toabout 25 amino acids in length) from a portion of RsGluCl or RsGluCl2 asdisclosed in SEQ ID NOs:2, 4, 6 and/or 8. Monospecific antibodies toRsGluCl are purified from mammalian antisera containing antibodiesreactive against RsGluCl or are prepared as monoclonal antibodiesreactive with RsGluCl using the technique of Kohler and Milstein (1975,Nature 256: 495-497). Monospecific antibody as used herein is defined asa single antibody species or multiple antibody species with homogenousbinding characteristics for RsGluCl. Homogenous binding as used hereinrefers to the ability of the antibody species to bind to a specificantigen or epitope, such as those associated with RsGluCl, as describedabove. Human RsGluCl-specific antibodies are raised by immunizinganimals such as mice, rats, guinea pigs, rabbits, goats, horses and thelike, with an appropriate concentration of RsGluCl protein or asynthetic peptide generated from a portion of RsGluCl with or without animmune adjuvant.

[0158] Preimmune serum is collected prior to the first immunization.Each animal receives between about 0.1 mg and about 1000 mg of RsGluClprotein associated with an acceptable immune adjuvant. Such acceptableadjuvants include, but are not limited to, Freund's complete, Freund'sincomplete, alum-precipitate, water in oil emulsion containingCorynebacterium parvum and tRNA. The initial immunization consists ofRsGluCl protein or peptide fragment thereof in, preferably, Freund'scomplete adjuvant at multiple sites either subcutaneously (SC),intraperitoneally (IP) or both. Each animal is bled at regularintervals, preferably weekly, to determine antibody titer. The animalsmay or may not receive booster injections following the initialimmunization. Those animals receiving booster injections are generallygiven an equal amount of RsGluCl in Freund's incomplete adjuvant by thesame route. Booster injections are given at about three week intervalsuntil maximal titers are obtained. At about 7 days after each boosterimmunization or about weekly after a single immunization, the animalsare bled, the serum collected, and aliquots are stored at about −20° C.

[0159] Monoclonal antibodies (mAb) reactive with RsGluCl are prepared byimmunizing inbred mice, preferably Balb/c, with RsGluCl protein. Themice are immunized by the IP or SC route with about 1 mg to about 100mg, preferably about 10 mg, of RsGluCl protein in about 0.5 ml buffer orsaline incorporated in an equal volume of an acceptable adjuvant, asdiscussed above. Freund's complete adjuvant is preferred. The micereceive an initial immunization on day 0 and are rested for about 3 toabout 30 weeks. Immunized mice are given one or more boosterimmunizations of about 1 to about 100 mg of RsGluCl in a buffer solutionsuch as phosphate buffered saline by the intravenous (IV) route.Lymphocytes, from antibody positive mice, preferably spleniclymphocytes, are obtained by removing spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions which will allow theformation of stable hybridomas. Fusion partners may include, but are notlimited to: mouse myelomas P3/NS 1/Ag 4-1; MPC-11; S-194 and Sp 2/0,with Sp 2/0 being preferred. The antibody producing cells and myelomacells are fused in polyethylene glycol, about 1000 mol. wt., atconcentrations from about 30% to about 50%. Fused hybridoma cells areselected by growth in hypoxanthine, thymidine and aminopterinsupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected form growth positivewells on about days 14, 18, and 21 and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay(SPIRA) using RsGluCl as the antigen. The culture fluids are also testedin the Ouchterlony precipitation assay to determine the isotype of themAb. Hybridoma cells from antibody positive wells are cloned by atechnique such as the soft agar technique of MacPherson, 1973, Soft AgarTechniques, in Tissue Culture Methods and Applications, Kruse andPaterson, Eds., Academic Press.

[0160] Monoclonal antibodies are produced in vivo by injection ofpristine primed Balb/c mice, approximately 0.5 ml per mouse, with about2×10⁶ to about 6×10⁶ hybridoma cells about 4 days after priming. Ascitesfluid is collected at approximately 8-12 days after cell transfer andthe monoclonal antibodies are purified by techniques known in the art.

[0161] In vitro production of anti-RsGluCl mAb is carried out by growingthe hybridoma in DMEM containing about 2% fetal calf serum to obtainsufficient quantities of the specific mAb. The mAb are purified bytechniques known in the art.

[0162] Antibody titers of ascites or hybridoma culture fluids aredetermined by various serological or immunological assays which include,but are not limited to, precipitation, passive agglutination,enzyme-linked immunosorbent antibody (ELISA) technique andradioimmunoassay (RIA) techniques. Similar assays are used to detect thepresence of RsGluCl in body fluids or tissue and cell extracts.

[0163] It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for RsGluCl peptide fragments, or arespective full-length RsGluCl.

[0164] RsGluCl antibody affinity columns are made, for example, byadding the antibodies to Affigel-10 (Biorad), a gel support which ispre-activated with N-hydroxysuccinimide esters such that the antibodiesform covalent linkages with the agarose gel bead support. The antibodiesare then coupled to the gel via amide bonds with the spacer arm. Theremaining activated esters are then quenched with 1M ethanolamine HCl(pH 8). The column is washed with water followed by 0.23 M glycine HCl(pH 2.6) to remove any non-conjugated antibody or extraneous protein.The column is then equilibrated in phosphate buffered saline (pH 7.3)and the cell culture supernatants or cell extracts containingfull-length RsGluCl or RsGluCl protein fragments are slowly passedthrough the column. The column is then washed with phosphate bufferedsaline until the optical density (A₂₈₀) falls to background, then theprotein is eluted with 0.23 M glycine-HCl (pH 2.6). The purified RsGluClprotein is then dialyzed against phosphate buffered saline.

[0165] The present invention also relates to a non-human transgenicanimal which is useful for studying the ability of a variety ofcompounds to act as modulators of RsGluCl, or any alternative functionalRsGluCl channel in vivo by providing cells for culture, in vitro. Inreference to the transgenic animals of this invention, reference is madeto transgenes and genes. As used herein, a transgene is a geneticconstruct including a gene. The transgene is integrated into one or morechromosomes in the cells in an animal by methods known in the art. Onceintegrated, the transgene is carried in at least one place in thechromosomes of a transgenic animal. Of course, a gene is a nucleotidesequence that encodes a protein, such as one or a combination of thecDNA clones described herein. The gene and/or transgene may also includegenetic regulatory elements and/or structural elements known in the art.A type of target cell for transgene introduction is the embryonic stemcell (ES). ES cells can be obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al., 1981, Nature292:154-156; Bradley et al., 1984, Nature 309:255-258; Gossler et al.,1986, Proc. Natl. Acad. Sci. USA 83:9065-9069; and Robertson et al.,1986 Nature 322:445448). Transgenes can be efficiently introduced intothe ES cells by a variety of standard techniques such as DNAtransfection, microinjection, or by retrovirus-mediated transduction.The resultant transformed ES cells can thereafter be combined withblastocysts from a non-human animal. The introduced ES cells thereaftercolonize the embryo and contribute to the germ line of the resultingchimeric animal (Jaenisch, 1988, Science 240: 1468-1474). It will alsobe within the purview of the skilled artisan to produce transgenic orknock-out invertebrate animals (e.g., C. elegans) which express theRsGluCl transgene in a wild type C. elegans GluCl background as well inC. elegans mutants knocked out for one or both of the C. elegans GluClsubunits.

[0166] Pharmaceutically useful compositions comprising modulators ofRsGluCl may be formulated according to known methods such as by theadmixture of a pharmaceutically acceptable carrier. Examples of suchcarriers and methods of formulation may be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the protein, DNA, RNA, modifiedRsGluCl, or either RsGluCl agonists or antagonists including tyrosinekinase activators or inhibitors.

[0167] Therapeutic or diagnostic compositions of the invention areadministered to an individual in amounts sufficient to treat or diagnosedisorders. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration.

[0168] The pharmaceutical compositions may be provided to the individualby a variety of routes such as subcutaneous, topical, oral andintramuscular.

[0169] The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

[0170] Compounds identified according to the methods disclosed hereinmay be used alone at appropriate dosages. Alternatively,co-administration or sequential administration of other agents may bedesirable.

[0171] The present invention also has the objective of providingsuitable topical, oral, systemic and parenteral pharmaceuticalformulations for use in the novel methods of treatment of the presentinvention. The compositions containing compounds identified according tothis invention as the active ingredient can be administered in a widevariety of therapeutic dosage forms in conventional vehicles foradministration. For example, the compounds can be administered in suchoral dosage forms as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, solutions, suspensions, syrups and emulsions, or byinjection. Likewise, they may also be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, or intramuscular form, all using forms well known tothose of ordinary skill in the pharmaceutical arts.

[0172] Advantageously, compounds of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.Furthermore, compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles, or viatransdermal routes, using those forms of transdermal skin patches wellknown to those of ordinary skill in that art. To be administered in theform of a transdermal delivery system, the dosage administration will,of course, be continuous rather than intermittent throughout the dosageregimen.

[0173] For combination treatment with more than one active agent, wherethe active agents are in separate dosage formulations, the active agentscan be administered concurrently, or they each can be administered atseparately staggered times.

[0174] The dosage regimen utilizing the compounds of the presentinvention is selected in accordance with a variety of factors includingtype, species, age, weight, sex and medical condition of the patient;the severity of the condition to be treated; the route ofadministration; the renal, hepatic and cardiovascular function of thepatient; and the particular compound thereof employed. A physician orveterinarian of ordinary skill can readily determine and prescribe theeffective amount of the drug required to prevent, counter or arrest theprogress of the condition. Optimal precision in achieving concentrationsof drug within the range that yields efficacy without toxicity requiresa regimen based on the kinetics of the drug's availability to targetsites. This involves a consideration of the distribution, equilibrium,and elimination of a drug.

[0175] The following examples are provided to illustrate the presentinvention without, however, limiting the same hereto.

EXAMPLE 1 Isolation and Characterization of DNA Molecules EncodingRsGluCl and RsGluCl2

[0176] Most molecular procedures were performed following standardprocedures available in references such as Ausubel et. al. (1992. Shortprotocols in molecular biology. F. M. Ausubel et al.,—2^(nd). ed. (JohnWiley & Sons), and Sambrook et al. (1989. Molecular cloning. Alaboratory manual. J. Sambrook, E. F. Fritsch, and T. Maniatis—2^(nd)ed. (Cold Spring Harbor Laboratory Press).

[0177] RsGluCl1

[0178] Adult brown dog tick polyA⁺ RNA was isolated using thePoly(A)Pure™ mRNA Isolation Kit (Ambion). Tick cDNA was synthesizedusing oligo-dT primers and the ZAP cDNA® Synthesis Kit (Stratagene),andcDNA>1 kb was selected using cDNA Size Fractionation Columns (BRL). Atick cDNA library was constructed in the Lambda ZAP® II vector using theGIGAPACK® III Gold Cloning Kit (Stratagene). A Drosophila GluCl cDNAfragment spanning the M1 to M3 region was used in a low-stringencyscreen [25% v/v formamide/5× SSCP (1×SSCP=120 mM NaCl/15 mM sodiumcitrate/20 mM sodium phosphate, pH 6.8)/0.1% SDS/10× Denhardt'ssolution/salmon sperm DNA (250 μg/ml) at 42° C.; wash, 0.2× SSC/0.1% SDSat 42° C.] of the tick cDNA library. The nucleotide sequence of theprobe is as follows: (SEQ ID NO:12)5′`ATTACTTAATACAAATTTATATACCATGCTGTATGTTGGTCATTGTATCATGGGTATCATTCTGGCTGGATCAAGGAGCAGTACCGGCGCGAGTGTCACTGGGTGTCACCACCCTGCTGACCATGGCCACCCAGACGTCGGGCATAAACGCCTCCCTGCCGCCCGTTTCCTATACGAAGGCCATCGATGTGTGGACAGGCGTGTGTCTGACGTTCGTGTTCGGGGCCCTGCTCGAGTTCGCCCTGGT G-3′.

[0179] Filters were exposed for eleven days and six positives wereisolted for sequence analysis. Three of the clones (T12, T82 and T32)encode GluCl-related proteins and were sequenced on both strands.

[0180] RsGluCl2

[0181] Poly (A)⁺ RNA was isolated from brown dog tick heads. Firststrand cDNA was synthesized from 50 ng RNA using a SUPERSCRIPTpreamplification System (Life Technologies). A tenth of the first strandreaction was used for PCR. The degenerate oligos utilized were designedbased on sequences obtained from C. elegans, Drosophila, and flea (C.felis) GluCls: Forward (27F2):GGAT(G/T)CCNGA(C/T)N(C/T)NTT(C/T)TTNN(A/C)NA(A/C)(C/T)G; (SEQ ID NO:9)Reverse 1 (3AF1): CNA(A/G)(A/C)A(A/G)NGCNC(A/C)GAANA(C/T)(A/G)AA(C/T)G;(SEQ ID NO:10) Reverse 2 (3AF2):CAN(A/G)CNCCN(A/G)(G/T)CCANAC(A/G)TCNA(C/T)N(A/G)C. (SEQ ID NO:11)

[0182] Two PCR rounds, using the combinations “27F2+3AF1, then27F2+3BF2” were performed. The cycles were as follow: 1× (95° C. for 120sec.), then 30× (95° C. for 45 sec.; 50° C. for 90 sec.; and 72° C. for120 sec.), then 1× (72° C. for 120 sec.). Reagents were from LifeTechnology Inc. The oligonucleotide concentration was 5 μM. One tenth ofthe PCR reaction products was tested by Southern blot analysis, in orderto identify and prevent the PCR-cloning of contaminating sequences.Novel PCR products of the appropriate size were cloned into the PCR2.1plasmid vector using a “TA” cloning kit (Invitrogen, Inc.). Followingsequence analysis (ABI Prism, PE Applied Biosystems), selected PCR cloneinserts were radiolabelled and used as probes to screen a cDNA librarygenerated into the Uni-ZAP® vector (Stratagene, Inc.) from using the RNApreparation mentioned above. Sequences from full-length cDNA clones wereanalysed using the GCG Inc. package. Subcloning of RsGluCl2 into amammalian expression vector was done by excision of an 1.85 kbcoding-region-containing fragment (XhoI-EcoRI digest) from the originalinsert of clone RsGluCl2 B1 from the UniZap® pBS plasmid, followed byligation into the TetSplice® vector (Life Technologies Inc.). cDNAclones T12 and T82 are identical in the coding region except for asingle nucleotide difference resulting in a single amino acidsubstitution which is probably a naturally ocurring polymorphism. TheT32 clone has 2 additional exons not present in the T12 and T82 cDNAs,one is near the 5′ end of the coding region (135 bp exon) and the otheris in the M3-M4 intracellular linker (96 bp exon). Additionally, theseoptional exons are not included in DrosGluCl-1 ORF. These cDNA clonesare also denoted as RsGluCl-1L (T32-2.48 kb) and RsGluCl-1S (T12 andT82-2.126 kb). The predicted RsGluCl-1S protein is approximately 71%identical to the DrosGluCl1 protein.

EXAMPLE 2 Functional Expression of RsGluCl1 and RsGluCl2 Clones inXenopus Oocytes

[0183]Xenopus laevis oocytes were prepared and injected using standardmethods previously described [Arena, J. P., Liu, K. K., Paress, P. S. &Cully, D. F. Mol. Pharmacol. 40, 368-374 (1991); Arena, J. P., Liu, K.K., Paress, P. S., Schaeffer, J. M. & Cully, D. F., Mol. Brain Res. 15,339-348 (1992)]. Adult female Xenopus laevis were anesthetized with0.17% tricaine methanesulfonate and the ovaries were surgically removedand placed in a solution consisting of (mM): NaCl 82.5, KCl 2, MgCl₂ 1,HEPES 5, NaPyruvate 2.5, Penicillin G. 100,000 units/L, StreptomycinSulfate 1000 mg/L, pH 7.5 (Mod. OR-2). Ovarian lobes were broken open,rinsed several times in Mod. OR-2, and incubated in 0.2% collagenase(Sigma, Type1) in Mod. OR-2 at room temperature with gentle shaking.After 1 hour the collagenase solution was renewed and the oocytes wereincubated for an additional 30-90 min until approximately 50% of theoocytes were released from the ovaries. Stage V and VI oocytes wereselected and placed in media containing (mM): NaCl 96, KCl 2, MgCl₂ 1,CaCl₂ 1.8, HEPES 5, NaPyruvate 2.5, theophylline 0.5, gentamicin 50mg/ml, pH 7.5 (ND-96) for 16-24 hours before injection. Oocytes wereinjected with 50 nl of Dv8, Dv9, RsGluCl or RsGluCl2 RNA at aconcentration of 0.2 mg/ml. Oocytes were incubated at 18° C. for 1-6days in ND-96 before recording.

[0184] Recordings were made at room temperature in modified ND-96consisting of (mM): NaCl 96, MgCl₂ 1, CaCl₂ 0.1, BaCl₂ 3.5, HEPES 5, pH7.5. Oocytes were voltage clamped using a Dagan CA1 two microelectrodeamplifier (Dagan Corporation, Minneapolis, Minn.) interfaced to aMacintosh 7100/80 computer. The current passing electrode was filledwith 0.7 M KCl, 1.7 M KCitrate, and the voltage recording electrode wasfilled with 1 M KCl. Throughout the experiment oocytes were superfusedwith modified ND-96 (control solution) or with ND-96 containingpotential channel activators and blockers at a rate of approximately 3ml/min. Data were acquired at 100 Hz and filtered at 33.3 Hz using Pulsesoftware from HEKA Elektronik (Lambrecht, Germany). All recordings wereperformed from a holding potential of either 0 or −30 mV.

[0185] cRNA was synthesized from the RsGluCl 1S clone T12 and expessedin Xenopus oocytes. The channel encoded by RsGluCl-1 is aglutamate-gated chloride channel activated by IVM-PO₄.

[0186]FIG. 10 shows the glutamate-activated current in oocytes injectedwith RsGluCl1 T12 RNA. Current activation was maximal with 10 μMglutamate and no current was seen in uninjected oocytes. Application of100 nM ivermectin produces a similar although non-inactivating current.

[0187]FIG. 11 shows the activation by ivermectin of RsGluCl2 expressedin Xenopus oocytes. Current activation was maximal with ˜1 μM ivermectinand glutamate failed to activate a current when expressed as a singlefunctional channel.

EXAMPLE 3 Functional Expression of RsGluCls Clones in Mammalian Cells

[0188] A RsGluCl may be subcloned into a mammalian expression vector andused to transfect the mammalian cell line of choice. Stable cell clonesare selected by growth in the presence of G418. Single G418 resistantclones are isolated and tested to confirm the presence of an intactRsGluCl gene. Clones containing the RsGluCls are then analyzed forexpression using immunological techniques, such as immuneprecipitation,Western blot, and immunofluorescence using antibodies specific to theRsGluCl proteins. Antibody is obtained from rabbits innoculated withpeptides that are synthesized from the amino acid sequence predictedfrom the RsGluCl sequences. Expression is also analyzed using patchclamp electrophysiological techniques and an anion flux assay.

[0189] Cells that are expressing RsGluCl stably or transiently, are usedto test for expression of active channel proteins. These cells are usedto identify and examine other compounds for their ability to modulate,inhibit or activate the respective channel.

[0190] Cassettes containing the RsGluCl cDNA in the positive orientationwith respect to the promoter are ligated into appropriate restrictionsites 3′ of the promoter and identified by restriction site mappingand/or sequencing. These cDNA expression vectors may be introduced intofibroblastic host cells, for example, COS-7 (ATCC# CRL1651), and CV-1tat [Sackevitz et al., 1987, Science 238: 1575], 293, L (ATCC# CRL6362)by standard methods including but not limited to electroporation, orchemical procedures (cationic liposomes, DEAE dextran, calciumphosphate). Transfected cells and cell culture supernatants can beharvested and analyzed for RsGluCl expression as described herein.

[0191] All of the vectors used for mammalian transient expression can beused to establish stable cell lines expressing RsGluCl. UnalteredRsGluCl cDNA constructs cloned into expression vectors are expected toprogram host cells to make RsGluCl protein. In addition, RsGluCl isexpressed extracellularly as a secreted protein by ligating RsGluCl cDNAconstructs to DNA encoding the signal sequence of a secreted protein.The transfection host cells include, but are not limited to, CV-1-P[Sackevitz et al., 1987, Science 238: 1575], tk-L [Wigler, et al., 1977,Cell 11: 223 ], NS/0, and dHFr- CHO [Kaufman and Sharp, 1982, J. Mol.Biol. 159: 601].

[0192] Co-transfection of any vector containing a RsGluCl cDNA with adrug selection plasmid including, but not limited to G418,aminoglycoside phosphotransferase; hygromycin, hygromycin-Bphosphotransferase; APRT, xanthine-guanine phosphoribosyl-transferase,will allow for the selection of stably transfected clones. Levels ofRsGluCl are quantitated by the assays described herein. RsGluCl cDNAconstructs may also be ligated into vectors containing amplifiabledrug-resistance markers for the production of mammalian cell clonessynthesizing the highest possible levels of RsGluCl. Followingintroduction of these constructs into cells, clones containing theplasmid are selected with the appropriate agent, and isolation of anover-expressing clone with a high copy number of plasmids isaccomplished by selection with increasing doses of the agent. Theexpression of recombinant RsGluCl is achieved by transfection offull-length RsGluCl cDNA into a mammalian host cell.

EXAMPLE 4 Cloning of RsGluCl cDNA into a Baculovirus Expression Vectorfor Expression in Insect Cells

[0193] Baculovirus vectors, which are derived from the genome of theAcNPV virus, are designed to provide high level expression of cDNA inthe Sf9 line of insect cells (ATCC CRL# 1711). A recombinantbaculoviruse expressing RsGluCl cDNA is produced by the followingstandard methods (In Vitrogen Maxbac Manual): The RsGluCl cDNAconstructs are ligated into the polyhedrin gene in a variety ofbaculovirus transfer vectors, including the pAC360 and the BlueBacvector (In Vitrogen). Recombinant baculoviruses are generated byhomologous recombination following co-transfection of the baculovirustransfer vector and linearized AcNPV genomic DNA [Kitts, 1990, Nuc.Acid. Res. 18: 5667] into Sf9-cells. Recombinant pAC360 viruses areidentified by the absence of inclusion bodies in infected cells andrecombinant pBlueBac viruses are identified on the basis ofb-galactosidase expression (Summers, M. D. and Smith, G. E., TexasAgriculture Exp. Station Bulletin No. 1555). Following plaquepurification, RsGluCl expression is measured by the assays describedherein.

[0194] The cDNA encoding the entire open reading frame for RsGluCl GluClis inserted into the BamHI site of pBlueBacII. Constructs in thepositive orientation are identified by sequence analysis and used totransfect Sf9 cells in the presence of linear AcNPV mild type DNA.

EXAMPLE 5 Cloning of RsGluCl cDNA into a Yeast Expression Vector

[0195] Recombinant RsGluCl is produced in the yeast S. cerevisiaefollowing the insertion of the optimal RsGluCl cDNA cistron intoexpression vectors designed to direct the intracellular or extracellularexpression of heterologous proteins. In the case of intracellularexpression, vectors such as EmBLyex4 or the like are ligated to theRsGluCl cistron [Rinas, et al., 1990, Biotechnology 8: 543-545; HorowitzB. et al., 1989, J. Biol. Chem. 265: 4189-4192]. For extracellularexpression, the RsGluCl GluCl cistron is ligated into yeast expressionvectors which fuse a secretion signal (a yeast or mammalian peptide) tothe NH₂ terminus of the RsGluCl protein [Jacobson, 1989, Gene 85:511-516; Riett and Bellon, 1989, Biochem. 28: 2941-2949].

[0196] These vectors include, but are not limited to pAVE1-6, whichfuses the human serum albumin signal to the expressed cDNA [Steep, 1990,Biotechnology 8: 42-46], and the vector pL8PL which fuses the humanlysozyme signal to the expressed cDNA [Yamamoto, Biochem. 28:2728-2732)]. In addition, RsGluCl is expressed in yeast as a fusionprotein conjugated to ubiquitin utilizing the vector pVEP [Ecker, 1989,J. Biol. Chem. 264: 7715-7719, Sabin, 1989 Biotechnology 7: 705-709,McDonnell, 1989, Mol. Cell Biol. 9: 5517-5523 (1989)]. The levels ofexpressed RsGluCl are determined by the assays described herein.

EXAMPLE 6 Purification of Recombinant RsGluCl

[0197] Recombinantly produced RsGluCl may be purified by antibodyaffinity chromatography. RsGluCl GluCl antibody affinity columns aremade by adding the anti-RsGluCl GluCl antibodies to Affigel-10 (Biorad),a gel support which is pre-activated with N-hydroxysuccinimide esterssuch that the antibodies form covalent linkages with the agarose gelbead support. The antibodies are then coupled to the gel via amide bondswith the spacer arm. The remaining activated esters are then quenchedwith 1M ethanolamine HCl (pH 8). The column is washed with waterfollowed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugatedantibody or extraneous protein. The column is then equilibrated inphosphate buffered saline (pH 7.3) together with appropriate membranesolubilizing agents such as detergents and the cell culture supernatantsor cell extracts containing solubilized RsGluCl are slowly passedthrough the column. The column is then washed with phosphate-bufferedsaline together with detergents until the optical density (A280) fallsto background, then the protein is eluted with 0.23 M glycine-HCl (pH2.6) together with detergents. The purified RsGluCl protein is thendialyzed against phosphate buffered saline.

1 12 1 2138 DNA Rhipicephalus sanguineus CDS (331)...(1683) 1 cgctcccccaatcctgaggt tccttctaac gagaaggagg agccacagcg ccggctgcgg 60 taccgccgcacgggccaacg tgagaccgcc cgagcccggc gccctgactt aggccgctga 120 gcgaaacccaaggcggcgcg ctggccactc cacgggaacg agaccggccc cctggagacg 180 acatcgtcgaccacaatgaa ctacttctct gacgtggcga agatggtggc ttcatcgaag 240 agagaaatcatcgaagcttt ccacgcgaca tctggagtac acggcgcatg cgaatgagcg 300 aacatcgctgaccgagactc gcccgtcacc atg agc gta cat tca tgg cgc ttt 354 Met Ser ValHis Ser Trp Arg Phe 1 5 tgt gtc cca ctg gtg gct cta gcg ttt ttc ttg ttgatt ctt ctg tcg 402 Cys Val Pro Leu Val Ala Leu Ala Phe Phe Leu Leu IleLeu Leu Ser 10 15 20 tgt cca tcg gca tgg ggc aag gca aat ttc cgc gct atagaa aag cgg 450 Cys Pro Ser Ala Trp Gly Lys Ala Asn Phe Arg Ala Ile GluLys Arg 25 30 35 40 ata ttg gac agc atc att ggc cag ggt cgt tat gac tgcagg atc cgg 498 Ile Leu Asp Ser Ile Ile Gly Gln Gly Arg Tyr Asp Cys ArgIle Arg 45 50 55 ccc atg gga att aac aac aca gac ggg ccg gct ctt gta cgcgtt aac 546 Pro Met Gly Ile Asn Asn Thr Asp Gly Pro Ala Leu Val Arg ValAsn 60 65 70 atc ttt gta aga agt atc ggc aga att gat gac gtc acc atg gagtac 594 Ile Phe Val Arg Ser Ile Gly Arg Ile Asp Asp Val Thr Met Glu Tyr75 80 85 aca gtg caa atg acg ttc aga gag cag tgg cgg gac gag aga ctc cag642 Thr Val Gln Met Thr Phe Arg Glu Gln Trp Arg Asp Glu Arg Leu Gln 9095 100 tac gac gac ttg ggc ggc cag gtt cgc tac ctg acg ctc acc gaa ccg690 Tyr Asp Asp Leu Gly Gly Gln Val Arg Tyr Leu Thr Leu Thr Glu Pro 105110 115 120 gac aag ctt tgg aag ccg gac ctg ttt ttc tcc aac gag aaa gaggga 738 Asp Lys Leu Trp Lys Pro Asp Leu Phe Phe Ser Asn Glu Lys Glu Gly125 130 135 cac ttc cac aac atc atc atg ccc aac gtg ctt cta cgc ata catccc 786 His Phe His Asn Ile Ile Met Pro Asn Val Leu Leu Arg Ile His Pro140 145 150 aac ggc gac gtt ctc ttc agc atc aga ata tcc ttg gtg ctt tcatgt 834 Asn Gly Asp Val Leu Phe Ser Ile Arg Ile Ser Leu Val Leu Ser Cys155 160 165 ccg atg aac ctg aaa ttt tat cct ttg gat aaa caa atc tgc tctatc 882 Pro Met Asn Leu Lys Phe Tyr Pro Leu Asp Lys Gln Ile Cys Ser Ile170 175 180 gtc atg gtg agc tat ggg tat aca aca gag gac ctg gtg ttt ctatgg 930 Val Met Val Ser Tyr Gly Tyr Thr Thr Glu Asp Leu Val Phe Leu Trp185 190 195 200 aaa gag ggg gat cct gta cag gtc aca aaa aat ctc cac ttgcca cgt 978 Lys Glu Gly Asp Pro Val Gln Val Thr Lys Asn Leu His Leu ProArg 205 210 215 ttc acg ctg gaa agg ttt caa acc gac tac tgc acc agt cggacc aac 1026 Phe Thr Leu Glu Arg Phe Gln Thr Asp Tyr Cys Thr Ser Arg ThrAsn 220 225 230 act ggc gag tac agc tgc ttg cgc gtg gac ctg gtg ttc aagcgc gag 1074 Thr Gly Glu Tyr Ser Cys Leu Arg Val Asp Leu Val Phe Lys ArgGlu 235 240 245 ttc agc tac tac ctg atc cag atc tac atc ccg tgc tgc atgctg gtc 1122 Phe Ser Tyr Tyr Leu Ile Gln Ile Tyr Ile Pro Cys Cys Met LeuVal 250 255 260 atc gtg tcc tgg gtg tcg ttc tgg ctc gac ccc acc tcg atcccg gcg 1170 Ile Val Ser Trp Val Ser Phe Trp Leu Asp Pro Thr Ser Ile ProAla 265 270 275 280 cga gtg tcg ctg ggc gtc acc acc ctg ctc acc atg gccacg cag ata 1218 Arg Val Ser Leu Gly Val Thr Thr Leu Leu Thr Met Ala ThrGln Ile 285 290 295 tcg ggc atc aac gcc tcg ctg cct ccc gtt tcc tac accaag gcc att 1266 Ser Gly Ile Asn Ala Ser Leu Pro Pro Val Ser Tyr Thr LysAla Ile 300 305 310 gac gtg tgg acc ggc gtc tgt ctg acc ttc gta ttc ggcgcg ctc ctc 1314 Asp Val Trp Thr Gly Val Cys Leu Thr Phe Val Phe Gly AlaLeu Leu 315 320 325 gag ttc gcc ctg gtc aac tac gcc tcg cgg tca gat tcacgc cgg cag 1362 Glu Phe Ala Leu Val Asn Tyr Ala Ser Arg Ser Asp Ser ArgArg Gln 330 335 340 aac atg cag aag cag aag cag agg aaa tgg gag ctc gagccg ccc ctg 1410 Asn Met Gln Lys Gln Lys Gln Arg Lys Trp Glu Leu Glu ProPro Leu 345 350 355 360 gac tcg gac cac ctg gag gac ggc gcc acc acg ttcgcc atg agg ccg 1458 Asp Ser Asp His Leu Glu Asp Gly Ala Thr Thr Phe AlaMet Arg Pro 365 370 375 ctg gtg cac cac cac gga gag ctg cat gcc gac aagttg cgg cag tgc 1506 Leu Val His His His Gly Glu Leu His Ala Asp Lys LeuArg Gln Cys 380 385 390 gaa gtc cac atg aag acc ccc aag acg aac ctt tgcaag gcc tgg ctt 1554 Glu Val His Met Lys Thr Pro Lys Thr Asn Leu Cys LysAla Trp Leu 395 400 405 tcc agg ttt ccc acg cga tcc aaa cgc atc gac gtcgtc tcg cgg atc 1602 Ser Arg Phe Pro Thr Arg Ser Lys Arg Ile Asp Val ValSer Arg Ile 410 415 420 ttc ttt ccg ctc atg ttc gcc ctc ttc aac ctc gtctac tgg aca acc 1650 Phe Phe Pro Leu Met Phe Ala Leu Phe Asn Leu Val TyrTrp Thr Thr 425 430 435 440 tac ctc ttc cgg gaa gac gag gaa gac gag tgacagaacacgg acgccacgac 1703 Tyr Leu Phe Arg Glu Asp Glu Glu Asp Glu * 445450 agccgccatc cgacaccatc gtcactgcag gcacgcactc tgtcgcgcgc acacaccacg1763 aagaccggcg cgccaacgca cgatgcgcgt tggccgctga aaaacccggg agcggggcgg1823 tgggggaggc tatgccccgg cccctcgctc ctcatcctcc gtgcacgctc gaatcgtcat1883 cgccacagcc agaaaaaaaa aagataccgt gcgaaaagtg gcggcaacac aacgtcgacg1943 ccatcagcgc cgcccagagc tgcaagcggc tcccacatgg ttgccaccgc agcttcctct2003 acgacccttc atccccaccg gcaccagcta cgagaaaggg accttatttc gggccatccc2063 tacataggcg actgttgttt tcgcacgaaa gatctttacg cagctgatgc tgaaaaaaaa2123 aaaaaaaaaa aaaaa 2138 2 450 PRT Rhipicephalus sanguineus 2 Met SerVal His Ser Trp Arg Phe Cys Val Pro Leu Val Ala Leu Ala 1 5 10 15 PhePhe Leu Leu Ile Leu Leu Ser Cys Pro Ser Ala Trp Gly Lys Ala 20 25 30 AsnPhe Arg Ala Ile Glu Lys Arg Ile Leu Asp Ser Ile Ile Gly Gln 35 40 45 GlyArg Tyr Asp Cys Arg Ile Arg Pro Met Gly Ile Asn Asn Thr Asp 50 55 60 GlyPro Ala Leu Val Arg Val Asn Ile Phe Val Arg Ser Ile Gly Arg 65 70 75 80Ile Asp Asp Val Thr Met Glu Tyr Thr Val Gln Met Thr Phe Arg Glu 85 90 95Gln Trp Arg Asp Glu Arg Leu Gln Tyr Asp Asp Leu Gly Gly Gln Val 100 105110 Arg Tyr Leu Thr Leu Thr Glu Pro Asp Lys Leu Trp Lys Pro Asp Leu 115120 125 Phe Phe Ser Asn Glu Lys Glu Gly His Phe His Asn Ile Ile Met Pro130 135 140 Asn Val Leu Leu Arg Ile His Pro Asn Gly Asp Val Leu Phe SerIle 145 150 155 160 Arg Ile Ser Leu Val Leu Ser Cys Pro Met Asn Leu LysPhe Tyr Pro 165 170 175 Leu Asp Lys Gln Ile Cys Ser Ile Val Met Val SerTyr Gly Tyr Thr 180 185 190 Thr Glu Asp Leu Val Phe Leu Trp Lys Glu GlyAsp Pro Val Gln Val 195 200 205 Thr Lys Asn Leu His Leu Pro Arg Phe ThrLeu Glu Arg Phe Gln Thr 210 215 220 Asp Tyr Cys Thr Ser Arg Thr Asn ThrGly Glu Tyr Ser Cys Leu Arg 225 230 235 240 Val Asp Leu Val Phe Lys ArgGlu Phe Ser Tyr Tyr Leu Ile Gln Ile 245 250 255 Tyr Ile Pro Cys Cys MetLeu Val Ile Val Ser Trp Val Ser Phe Trp 260 265 270 Leu Asp Pro Thr SerIle Pro Ala Arg Val Ser Leu Gly Val Thr Thr 275 280 285 Leu Leu Thr MetAla Thr Gln Ile Ser Gly Ile Asn Ala Ser Leu Pro 290 295 300 Pro Val SerTyr Thr Lys Ala Ile Asp Val Trp Thr Gly Val Cys Leu 305 310 315 320 ThrPhe Val Phe Gly Ala Leu Leu Glu Phe Ala Leu Val Asn Tyr Ala 325 330 335Ser Arg Ser Asp Ser Arg Arg Gln Asn Met Gln Lys Gln Lys Gln Arg 340 345350 Lys Trp Glu Leu Glu Pro Pro Leu Asp Ser Asp His Leu Glu Asp Gly 355360 365 Ala Thr Thr Phe Ala Met Arg Pro Leu Val His His His Gly Glu Leu370 375 380 His Ala Asp Lys Leu Arg Gln Cys Glu Val His Met Lys Thr ProLys 385 390 395 400 Thr Asn Leu Cys Lys Ala Trp Leu Ser Arg Phe Pro ThrArg Ser Lys 405 410 415 Arg Ile Asp Val Val Ser Arg Ile Phe Phe Pro LeuMet Phe Ala Leu 420 425 430 Phe Asn Leu Val Tyr Trp Thr Thr Tyr Leu PheArg Glu Asp Glu Glu 435 440 445 Asp Glu 450 3 2289 DNA Rhipicephalussanguineus CDS (502)...(1854) 3 cacacctcct gcgtctctcc actcgatgaagacctgtccc ggaggcgcga gcccaactgc 60 gcgctctgtc cgcatgtgtc gccgccactgagaggcctcc ggcgtggcgc gcttgtcaac 120 gcggcgcgcc ggcccgcagc aaatcgcgggcattccactc agggtctcat tcgctccccc 180 aatcctgagg ttccttctaa cgagaaggaggagccacagc gccggctgcg gtaccgccgc 240 acgggccaac gtgagaccgc ccgagcccggcgccctgact taggccgctg agcgaaaccc 300 aaggcggcgc gctggccact ccacgggaacgagaccggcc ccctggagac gacatcgtcg 360 accacaatga actacttctc tgacgtggcgaagatggtgg cttcatcgaa gagagaaatc 420 atcgaagctt tccacgcgac atctggagtacacggcgcat gcgaatgagc gaacatcgct 480 gaccgagact cgcccgtcac c atg agc gtacat tca tgg cgc ttt tgt gtc 531 Met Ser Val His Ser Trp Arg Phe Cys Val1 5 10 cca ctg gtg gct cta gcg ttt ttc ttg ttg att ctt ctg tcg tgt cca579 Pro Leu Val Ala Leu Ala Phe Phe Leu Leu Ile Leu Leu Ser Cys Pro 1520 25 tcg gca tgg ggc aag gca aat ttc cgc gct ata gaa aag cgg ata ttg627 Ser Ala Trp Gly Lys Ala Asn Phe Arg Ala Ile Glu Lys Arg Ile Leu 3035 40 gac agc atc att ggc cag ggt cgt tat gac tgc agg atc cgg ccc atg675 Asp Ser Ile Ile Gly Gln Gly Arg Tyr Asp Cys Arg Ile Arg Pro Met 4550 55 gga att aac aac aca gac ggg ccg gct ctt gta cgc gtt aac atc ttt723 Gly Ile Asn Asn Thr Asp Gly Pro Ala Leu Val Arg Val Asn Ile Phe 6065 70 gta aga agt atc ggc aga att gat gac gtc acc atg gag tac aca gtg771 Val Arg Ser Ile Gly Arg Ile Asp Asp Val Thr Met Glu Tyr Thr Val 7580 85 90 caa atg acg ttc aga gag cag tgg cgg gac gag aga ctc cag tac gac819 Gln Met Thr Phe Arg Glu Gln Trp Arg Asp Glu Arg Leu Gln Tyr Asp 95100 105 gac ttg ggc ggc cag gtt cgc tac ctg acg ctc acc gaa ccg gac aag867 Asp Leu Gly Gly Gln Val Arg Tyr Leu Thr Leu Thr Glu Pro Asp Lys 110115 120 ctt tgg aag ccg gac ctg ttt ttc tcc aac gag aaa gag gga cac ttc915 Leu Trp Lys Pro Asp Leu Phe Phe Ser Asn Glu Lys Glu Gly His Phe 125130 135 cac aac atc atc atg ccc aac gtg ctt cta cgc ata cat ccc aac ggc963 His Asn Ile Ile Met Pro Asn Val Leu Leu Arg Ile His Pro Asn Gly 140145 150 gac gtt ctc ttc agc atc aga ata tcc ttg gtg ctt tca tgt ccg atg1011 Asp Val Leu Phe Ser Ile Arg Ile Ser Leu Val Leu Ser Cys Pro Met 155160 165 170 aac ctg aaa ttt tat cct ttg gat aaa caa atc tgc tct atc gtcatg 1059 Asn Leu Lys Phe Tyr Pro Leu Asp Lys Gln Ile Cys Ser Ile Val Met175 180 185 gtg agc tat ggg tat aca aca gag gac ctg gtg ttt cta tgg aaagag 1107 Val Ser Tyr Gly Tyr Thr Thr Glu Asp Leu Val Phe Leu Trp Lys Glu190 195 200 ggg gat cct gta cag gtc aca aaa aat ctc cac ttg cca cgt ttcacg 1155 Gly Asp Pro Val Gln Val Thr Lys Asn Leu His Leu Pro Arg Phe Thr205 210 215 ctg gaa agg ttt caa acc gac tac tgc acc agt cgg acc aac actggc 1203 Leu Glu Arg Phe Gln Thr Asp Tyr Cys Thr Ser Arg Thr Asn Thr Gly220 225 230 gag tac agc tgc ttg cgc gtg gac ctg gtg ttc aag cgc gag ttcagc 1251 Glu Tyr Ser Cys Leu Arg Val Asp Leu Val Phe Lys Arg Glu Phe Ser235 240 245 250 tac tac ctg atc cag atc tac atc ccg tgc tgc atg ctg gtcatc gtg 1299 Tyr Tyr Leu Ile Gln Ile Tyr Ile Pro Cys Cys Met Leu Val IleVal 255 260 265 tcc tgg gtg tcg ttc tgg ctc gac ccc acc tcg atc ccg gcgcga gtg 1347 Ser Trp Val Ser Phe Trp Leu Asp Pro Thr Ser Ile Pro Ala ArgVal 270 275 280 tcg ctg ggc gtc acc acc ctg ctc acc atg gcc acg cag atatcg ggc 1395 Ser Leu Gly Val Thr Thr Leu Leu Thr Met Ala Thr Gln Ile SerGly 285 290 295 atc aac gcc tcg ctg cct ccc gtt tcc tac acc aag gcc attgac gtg 1443 Ile Asn Ala Ser Leu Pro Pro Val Ser Tyr Thr Lys Ala Ile AspVal 300 305 310 tgg acc ggc gtc tgt ctg acc ttc gta ttc ggc gcg ctc ctcgag ttc 1491 Trp Thr Gly Val Cys Leu Thr Phe Val Phe Gly Ala Leu Leu GluPhe 315 320 325 330 gcc ctg gtc aac tac gcc tcg cgg tca gat tca cgc cggcag aac atg 1539 Ala Leu Val Asn Tyr Ala Ser Arg Ser Asp Ser Arg Arg GlnAsn Met 335 340 345 cag aag cag aag cag agg aaa tgg gag ctc gag ccg cccctg gac tcg 1587 Gln Lys Gln Lys Gln Arg Lys Trp Glu Leu Glu Pro Pro LeuAsp Ser 350 355 360 gac cac ctg gag gac ggc gcc acc acg ttc gcc atg aggccg ctg gtg 1635 Asp His Leu Glu Asp Gly Ala Thr Thr Phe Ala Met Arg ProLeu Val 365 370 375 cac cac cac gga gag ctg cat gcc gac aag ttg cgg cagtgc gaa gtc 1683 His His His Gly Glu Leu His Ala Asp Lys Leu Arg Gln CysGlu Val 380 385 390 cac atg aag acc ccc aag acg aac ctt tgc aag gcc tggctt tcc agg 1731 His Met Lys Thr Pro Lys Thr Asn Leu Cys Lys Ala Trp LeuSer Arg 395 400 405 410 ttt ccc acg cga tcc aaa cgc atc gac gtc gtc tcgcgg atc ttc ttt 1779 Phe Pro Thr Arg Ser Lys Arg Ile Asp Val Val Ser ArgIle Phe Phe 415 420 425 ccg ctc atg ttc gcc ctc ttc aac ctc gtc tac tggaca acc tac ctc 1827 Pro Leu Met Phe Ala Leu Phe Asn Leu Val Tyr Trp ThrThr Tyr Leu 430 435 440 ttc cgg gaa gac aag gaa gac gag tga cagaacacgaacgccacgac 1874 Phe Arg Glu Asp Lys Glu Asp Glu * 445 450 agccgccatccgacaccatc gtcactgcag gcacgcactc tgtcgcgcgc acacaccacg 1934 aagaccggcgcgccaacgca cgatgcgcgt tggccgctga aaaacccggg agcggggcgg 1994 tgggggaggctatgccccgg cccctcgctc ctcatcctcc gtgcacgctc gaatcgtcat 2054 cgccacagccagaaaaaaaa aagataccgt gcgaaaagtg gcggcaacac aacgtcgacg 2114 ccatcagcgccgcccagagc tgcaagcggc tcccacatgg ttgccaccgc agcttcctct 2174 acgacccttcatccccaccg gcaccagcta cgagaaaggg accttatttc gggccatccc 2234 tacataggcgactgttgttt tcgcacgaaa gatctttacg cagctgatgc tgaaa 2289 4 450 PRTRhipicephalus sanguineus 4 Met Ser Val His Ser Trp Arg Phe Cys Val ProLeu Val Ala Leu Ala 1 5 10 15 Phe Phe Leu Leu Ile Leu Leu Ser Cys ProSer Ala Trp Gly Lys Ala 20 25 30 Asn Phe Arg Ala Ile Glu Lys Arg Ile LeuAsp Ser Ile Ile Gly Gln 35 40 45 Gly Arg Tyr Asp Cys Arg Ile Arg Pro MetGly Ile Asn Asn Thr Asp 50 55 60 Gly Pro Ala Leu Val Arg Val Asn Ile PheVal Arg Ser Ile Gly Arg 65 70 75 80 Ile Asp Asp Val Thr Met Glu Tyr ThrVal Gln Met Thr Phe Arg Glu 85 90 95 Gln Trp Arg Asp Glu Arg Leu Gln TyrAsp Asp Leu Gly Gly Gln Val 100 105 110 Arg Tyr Leu Thr Leu Thr Glu ProAsp Lys Leu Trp Lys Pro Asp Leu 115 120 125 Phe Phe Ser Asn Glu Lys GluGly His Phe His Asn Ile Ile Met Pro 130 135 140 Asn Val Leu Leu Arg IleHis Pro Asn Gly Asp Val Leu Phe Ser Ile 145 150 155 160 Arg Ile Ser LeuVal Leu Ser Cys Pro Met Asn Leu Lys Phe Tyr Pro 165 170 175 Leu Asp LysGln Ile Cys Ser Ile Val Met Val Ser Tyr Gly Tyr Thr 180 185 190 Thr GluAsp Leu Val Phe Leu Trp Lys Glu Gly Asp Pro Val Gln Val 195 200 205 ThrLys Asn Leu His Leu Pro Arg Phe Thr Leu Glu Arg Phe Gln Thr 210 215 220Asp Tyr Cys Thr Ser Arg Thr Asn Thr Gly Glu Tyr Ser Cys Leu Arg 225 230235 240 Val Asp Leu Val Phe Lys Arg Glu Phe Ser Tyr Tyr Leu Ile Gln Ile245 250 255 Tyr Ile Pro Cys Cys Met Leu Val Ile Val Ser Trp Val Ser PheTrp 260 265 270 Leu Asp Pro Thr Ser Ile Pro Ala Arg Val Ser Leu Gly ValThr Thr 275 280 285 Leu Leu Thr Met Ala Thr Gln Ile Ser Gly Ile Asn AlaSer Leu Pro 290 295 300 Pro Val Ser Tyr Thr Lys Ala Ile Asp Val Trp ThrGly Val Cys Leu 305 310 315 320 Thr Phe Val Phe Gly Ala Leu Leu Glu PheAla Leu Val Asn Tyr Ala 325 330 335 Ser Arg Ser Asp Ser Arg Arg Gln AsnMet Gln Lys Gln Lys Gln Arg 340 345 350 Lys Trp Glu Leu Glu Pro Pro LeuAsp Ser Asp His Leu Glu Asp Gly 355 360 365 Ala Thr Thr Phe Ala Met ArgPro Leu Val His His His Gly Glu Leu 370 375 380 His Ala Asp Lys Leu ArgGln Cys Glu Val His Met Lys Thr Pro Lys 385 390 395 400 Thr Asn Leu CysLys Ala Trp Leu Ser Arg Phe Pro Thr Arg Ser Lys 405 410 415 Arg Ile AspVal Val Ser Arg Ile Phe Phe Pro Leu Met Phe Ala Leu 420 425 430 Phe AsnLeu Val Tyr Trp Thr Thr Tyr Leu Phe Arg Glu Asp Lys Glu 435 440 445 AspGlu 450 5 2400 DNA Rhipicephalus sanguineus CDS (617)...(2170) 5caggctccgg cgtgactgtc gctcgctcgg ctctcgacgc tcgcggcggg aacaaccgct 60acccggacgc tcgatcagga gcagttcggg ccacagagaa aggggccgag gagtgcacac 120ctcctgcgtc tctccactcg atgaagacct gtcccggagg cgcgagccca actgcgcgct 180ctgtccgcat gtgtcgccgc cactgagagg cctccggcgt ggcgcgcttg tcaacgcggc 240gcgccggccc gcagcaaatc gcgggcattc cactcagggt ctcattcgct cccccaatcc 300tgaggttcct tctaacgaga aggaggagcc acagcgccgg ctgcggtacc gccgcacggg 360ccaacgtgag accgcccgag cccggcgccc tgacttaggc cgctgagcga aacccaaggc 420ggcgcgctgg ccactccacg ggaacgagac cggccccctg gagacgacat cgtcgaccac 480aatgaactac ttctctgacg tggcgaagat ggtggcttca tcgaagagag aaatcatcga 540agctttccac gcgacatctg gagtacacgg cgcatgcgaa tgagcgaaca tcgctgaccg 600agactcgccc gtcacc atg agc gta cat tca tgg cgc ttt tgt gtc cca ctg 652Met Ser Val His Ser Trp Arg Phe Cys Val Pro Leu 1 5 10 gtg gct cta gcgttt ttc ttg ttg att ctt ctg tcg tgt cca tcg gca 700 Val Ala Leu Ala PhePhe Leu Leu Ile Leu Leu Ser Cys Pro Ser Ala 15 20 25 tgg gcc gaa acg ctgcct acg cca cca acc cgt ggc cag ggg ggc gtt 748 Trp Ala Glu Thr Leu ProThr Pro Pro Thr Arg Gly Gln Gly Gly Val 30 35 40 ccg gtc gcg gcc gcg atgctc ctg ggg aaa cag caa agt tcc cgc tac 796 Pro Val Ala Ala Ala Met LeuLeu Gly Lys Gln Gln Ser Ser Arg Tyr 45 50 55 60 caa gat aaa gag ggc aaggca aat ttc cgc gct ata gaa aag cgg ata 844 Gln Asp Lys Glu Gly Lys AlaAsn Phe Arg Ala Ile Glu Lys Arg Ile 65 70 75 ttg gac agc atc att ggc cagggt cgt tat gac tgc agg atc cgg ccc 892 Leu Asp Ser Ile Ile Gly Gln GlyArg Tyr Asp Cys Arg Ile Arg Pro 80 85 90 atg gga att aac aac aca gac gggccg gct ctt gta cgc gtt aac atc 940 Met Gly Ile Asn Asn Thr Asp Gly ProAla Leu Val Arg Val Asn Ile 95 100 105 ttt gta aga agt atc ggc aga attgat gac gtc acc atg gag tac aca 988 Phe Val Arg Ser Ile Gly Arg Ile AspAsp Val Thr Met Glu Tyr Thr 110 115 120 gtg caa atg acg ttc aga gag cagtgg cgg gac gag aga ctc cag tac 1036 Val Gln Met Thr Phe Arg Glu Gln TrpArg Asp Glu Arg Leu Gln Tyr 125 130 135 140 gac gac ttg ggc ggc cag gttcgc tac ctg acg ctc acc gaa ccg gac 1084 Asp Asp Leu Gly Gly Gln Val ArgTyr Leu Thr Leu Thr Glu Pro Asp 145 150 155 aag ctt tgg aag ccg gac ctgttt ttc tcc aac gag aaa gag gga cac 1132 Lys Leu Trp Lys Pro Asp Leu PhePhe Ser Asn Glu Lys Glu Gly His 160 165 170 ttc cac aac atc atc atg cccaac gtg ctt cta cgc ata cat ccc aac 1180 Phe His Asn Ile Ile Met Pro AsnVal Leu Leu Arg Ile His Pro Asn 175 180 185 ggc gac gtt ctc ttc agc atcaga ata tcc ttg gtg ctt tca tgt ccg 1228 Gly Asp Val Leu Phe Ser Ile ArgIle Ser Leu Val Leu Ser Cys Pro 190 195 200 atg aac ctg aaa ttt tat cctttg gat aaa caa atc tgc tct atc gtc 1276 Met Asn Leu Lys Phe Tyr Pro LeuAsp Lys Gln Ile Cys Ser Ile Val 205 210 215 220 atg gtg agc tat ggg tataca aca gag gac ctg gtg ttt cta tgg aaa 1324 Met Val Ser Tyr Gly Tyr ThrThr Glu Asp Leu Val Phe Leu Trp Lys 225 230 235 gag ggg gat cct gta caggtc aca aaa aat ctc cac ttg cca cgt ttc 1372 Glu Gly Asp Pro Val Gln ValThr Lys Asn Leu His Leu Pro Arg Phe 240 245 250 acg ctg gaa agg ttt caaacc gac tac tgc acc agt cgg acc aac act 1420 Thr Leu Glu Arg Phe Gln ThrAsp Tyr Cys Thr Ser Arg Thr Asn Thr 255 260 265 ggc gag tac agc tgc ttgcgc gtg gac ctg gtg ttc aag cgc gag ttc 1468 Gly Glu Tyr Ser Cys Leu ArgVal Asp Leu Val Phe Lys Arg Glu Phe 270 275 280 agc tac tac ctg atc cagatc tac atc ccg tgc tgc atg ctg gtc atc 1516 Ser Tyr Tyr Leu Ile Gln IleTyr Ile Pro Cys Cys Met Leu Val Ile 285 290 295 300 gtg tcc tgg gtg tcgttc tgg ctc gac ccc acc tcg atc ccg gcg cga 1564 Val Ser Trp Val Ser PheTrp Leu Asp Pro Thr Ser Ile Pro Ala Arg 305 310 315 gtg tcg ctg ggc gtcacc acc ctg ctc acc atg gcc acg cag ata tcg 1612 Val Ser Leu Gly Val ThrThr Leu Leu Thr Met Ala Thr Gln Ile Ser 320 325 330 ggc atc aac gcc tcgctg cct ccc gtt tcc tac acc aag gcc att gac 1660 Gly Ile Asn Ala Ser LeuPro Pro Val Ser Tyr Thr Lys Ala Ile Asp 335 340 345 gtg tgg acc ggc gtctgt ctg acc ttc gta ttc ggc gcg ctc ctc gag 1708 Val Trp Thr Gly Val CysLeu Thr Phe Val Phe Gly Ala Leu Leu Glu 350 355 360 ttc gcc ctg gtc aactac gcc tcg cgg tca gat tca cgc cgg cag aac 1756 Phe Ala Leu Val Asn TyrAla Ser Arg Ser Asp Ser Arg Arg Gln Asn 365 370 375 380 atg cag aag cagaag cag agg aaa tgg gag ctc gag ccg ccc ctg gac 1804 Met Gln Lys Gln LysGln Arg Lys Trp Glu Leu Glu Pro Pro Leu Asp 385 390 395 tcg gac cac ctggag gac ggc gcc acc acg ttc gcc atg gtg agc tcc 1852 Ser Asp His Leu GluAsp Gly Ala Thr Thr Phe Ala Met Val Ser Ser 400 405 410 ggc gag ccg gcgggc ctc atg gcg cga acc tgg cca cca ccg ccg ctg 1900 Gly Glu Pro Ala GlyLeu Met Ala Arg Thr Trp Pro Pro Pro Pro Leu 415 420 425 ccg cca aac atggcg gcc ggc tcc gcg caa gcc ggc gcc agg ccg ctg 1948 Pro Pro Asn Met AlaAla Gly Ser Ala Gln Ala Gly Ala Arg Pro Leu 430 435 440 gtg cac cac cacgga gag ctg cat gcc gac aag ttg cgg cag tgc gaa 1996 Val His His His GlyGlu Leu His Ala Asp Lys Leu Arg Gln Cys Glu 445 450 455 460 gtc cac atgaag acc ccc aag acg aac ctt tgc aag gcc tgg ctt tcc 2044 Val His Met LysThr Pro Lys Thr Asn Leu Cys Lys Ala Trp Leu Ser 465 470 475 agg ttt cccacg cga tcc aaa cgc atc gac gtc gtc tcg cgg atc ttc 2092 Arg Phe Pro ThrArg Ser Lys Arg Ile Asp Val Val Ser Arg Ile Phe 480 485 490 ttt ccg ctcgtg ttc gcc ctc ttc aac ctc gtc tac tgg aca acc tac 2140 Phe Pro Leu ValPhe Ala Leu Phe Asn Leu Val Tyr Trp Thr Thr Tyr 495 500 505 ctc ttc cgggaa gac gag gag gac gag tga cagaacacga acgccacgac 2190 Leu Phe Arg GluAsp Glu Glu Asp Glu * 510 515 agccgccatc cgacaccatc gtcactgcaggcacgcactc tgtcgcgcgc acacaccacg 2250 aagaccggcg cgccaacgca cgatgcgcgttggccgctga aaaacccggg agcggggcgg 2310 tgggggaggc tatgccccgg cccctcgctcctcatcctcc gtgcacgctc gaatcgtcat 2370 cgccacagcc agaaaaaaaa aaaaaaaaaa2400 6 517 PRT Rhipicephalus sanguineus 6 Met Ser Val His Ser Trp ArgPhe Cys Val Pro Leu Val Ala Leu Ala 1 5 10 15 Phe Phe Leu Leu Ile LeuLeu Ser Cys Pro Ser Ala Trp Ala Glu Thr 20 25 30 Leu Pro Thr Pro Pro ThrArg Gly Gln Gly Gly Val Pro Val Ala Ala 35 40 45 Ala Met Leu Leu Gly LysGln Gln Ser Ser Arg Tyr Gln Asp Lys Glu 50 55 60 Gly Lys Ala Asn Phe ArgAla Ile Glu Lys Arg Ile Leu Asp Ser Ile 65 70 75 80 Ile Gly Gln Gly ArgTyr Asp Cys Arg Ile Arg Pro Met Gly Ile Asn 85 90 95 Asn Thr Asp Gly ProAla Leu Val Arg Val Asn Ile Phe Val Arg Ser 100 105 110 Ile Gly Arg IleAsp Asp Val Thr Met Glu Tyr Thr Val Gln Met Thr 115 120 125 Phe Arg GluGln Trp Arg Asp Glu Arg Leu Gln Tyr Asp Asp Leu Gly 130 135 140 Gly GlnVal Arg Tyr Leu Thr Leu Thr Glu Pro Asp Lys Leu Trp Lys 145 150 155 160Pro Asp Leu Phe Phe Ser Asn Glu Lys Glu Gly His Phe His Asn Ile 165 170175 Ile Met Pro Asn Val Leu Leu Arg Ile His Pro Asn Gly Asp Val Leu 180185 190 Phe Ser Ile Arg Ile Ser Leu Val Leu Ser Cys Pro Met Asn Leu Lys195 200 205 Phe Tyr Pro Leu Asp Lys Gln Ile Cys Ser Ile Val Met Val SerTyr 210 215 220 Gly Tyr Thr Thr Glu Asp Leu Val Phe Leu Trp Lys Glu GlyAsp Pro 225 230 235 240 Val Gln Val Thr Lys Asn Leu His Leu Pro Arg PheThr Leu Glu Arg 245 250 255 Phe Gln Thr Asp Tyr Cys Thr Ser Arg Thr AsnThr Gly Glu Tyr Ser 260 265 270 Cys Leu Arg Val Asp Leu Val Phe Lys ArgGlu Phe Ser Tyr Tyr Leu 275 280 285 Ile Gln Ile Tyr Ile Pro Cys Cys MetLeu Val Ile Val Ser Trp Val 290 295 300 Ser Phe Trp Leu Asp Pro Thr SerIle Pro Ala Arg Val Ser Leu Gly 305 310 315 320 Val Thr Thr Leu Leu ThrMet Ala Thr Gln Ile Ser Gly Ile Asn Ala 325 330 335 Ser Leu Pro Pro ValSer Tyr Thr Lys Ala Ile Asp Val Trp Thr Gly 340 345 350 Val Cys Leu ThrPhe Val Phe Gly Ala Leu Leu Glu Phe Ala Leu Val 355 360 365 Asn Tyr AlaSer Arg Ser Asp Ser Arg Arg Gln Asn Met Gln Lys Gln 370 375 380 Lys GlnArg Lys Trp Glu Leu Glu Pro Pro Leu Asp Ser Asp His Leu 385 390 395 400Glu Asp Gly Ala Thr Thr Phe Ala Met Val Ser Ser Gly Glu Pro Ala 405 410415 Gly Leu Met Ala Arg Thr Trp Pro Pro Pro Pro Leu Pro Pro Asn Met 420425 430 Ala Ala Gly Ser Ala Gln Ala Gly Ala Arg Pro Leu Val His His His435 440 445 Gly Glu Leu His Ala Asp Lys Leu Arg Gln Cys Glu Val His MetLys 450 455 460 Thr Pro Lys Thr Asn Leu Cys Lys Ala Trp Leu Ser Arg PhePro Thr 465 470 475 480 Arg Ser Lys Arg Ile Asp Val Val Ser Arg Ile PhePhe Pro Leu Val 485 490 495 Phe Ala Leu Phe Asn Leu Val Tyr Trp Thr ThrTyr Leu Phe Arg Glu 500 505 510 Asp Glu Glu Asp Glu 515 7 1402 DNARhipicephalus sanguineus CDS (131)...(1385) 7 cgccgctcaa tcgcgggctacggactcgtc gttcccggag gggcttggac cacagctcgc 60 tcgtcaccgt ggtggctggccgcttcgcct ggcggtcctg cacgcacgct gtaacgaacg 120 tcgccacgcg atg ttt ggtgtg cca tgc tcc cgc gcc tgc cgc ctt gtg 169 Met Phe Gly Val Pro Cys SerArg Ala Cys Arg Leu Val 1 5 10 gtg gtg ata gct gcg ttc tgc tgg ccg cccgct ctg ccg ctc gta ccc 217 Val Val Ile Ala Ala Phe Cys Trp Pro Pro AlaLeu Pro Leu Val Pro 15 20 25 ggg gga gtt tcc tcc aga gca aac gat ctg gacatt ctg gac gag ctc 265 Gly Gly Val Ser Ser Arg Ala Asn Asp Leu Asp IleLeu Asp Glu Leu 30 35 40 45 ctc aaa aac tac gat cga agg gcc ctg ccg agcagt cac ctc gga aat 313 Leu Lys Asn Tyr Asp Arg Arg Ala Leu Pro Ser SerHis Leu Gly Asn 50 55 60 gca act att gtg tca tgc gaa att tac ata cga agtttt gga tca ata 361 Ala Thr Ile Val Ser Cys Glu Ile Tyr Ile Arg Ser PheGly Ser Ile 65 70 75 aat cct tcg aac atg gac tac gaa gtc gac ctc tac ttccgg cag tcg 409 Asn Pro Ser Asn Met Asp Tyr Glu Val Asp Leu Tyr Phe ArgGln Ser 80 85 90 tgg ctc gac gag cgg tta cgc aaa tcc acg cta tct cgt ccgctc gac 457 Trp Leu Asp Glu Arg Leu Arg Lys Ser Thr Leu Ser Arg Pro LeuAsp 95 100 105 ctt aat gac cca aag ctg gta caa atg ata tgg aag cca gaagtt ttc 505 Leu Asn Asp Pro Lys Leu Val Gln Met Ile Trp Lys Pro Glu ValPhe 110 115 120 125 ttt gcg aac gcg aaa cac gcc gag ttc caa tat gtg actgta cct aac 553 Phe Ala Asn Ala Lys His Ala Glu Phe Gln Tyr Val Thr ValPro Asn 130 135 140 gtc ctc gtt agg atc aac ccg act gga ata atc ttg tacatg ttg cgg 601 Val Leu Val Arg Ile Asn Pro Thr Gly Ile Ile Leu Tyr MetLeu Arg 145 150 155 tta aaa ctg agg ttc tcc tgc atg atg gac ctg tac cggtac ccc atg 649 Leu Lys Leu Arg Phe Ser Cys Met Met Asp Leu Tyr Arg TyrPro Met 160 165 170 gat tcc caa gtc tgc agc atc gaa att gcc tct ttt tccaaa acc acc 697 Asp Ser Gln Val Cys Ser Ile Glu Ile Ala Ser Phe Ser LysThr Thr 175 180 185 gaa gag ctg ctg ctg aaa tgg tcc gag agt cag cct gtcgtt ctc ttc 745 Glu Glu Leu Leu Leu Lys Trp Ser Glu Ser Gln Pro Val ValLeu Phe 190 195 200 205 gat aac ctc aag ttg ccc cag ttt gaa ata gag aaggtg aac acg tcc 793 Asp Asn Leu Lys Leu Pro Gln Phe Glu Ile Glu Lys ValAsn Thr Ser 210 215 220 tta tgc aaa gaa aag ttt cac ata ggg gaa tac agttgc ctg aaa gcc 841 Leu Cys Lys Glu Lys Phe His Ile Gly Glu Tyr Ser CysLeu Lys Ala 225 230 235 gac ttc tat ctg cag cgt tcc ctc ggt tat cac atggtg cag acc tat 889 Asp Phe Tyr Leu Gln Arg Ser Leu Gly Tyr His Met ValGln Thr Tyr 240 245 250 ctt ccg acc acg ctt atc gtg gtc atc tca tgg gtgtca ttc tgg ctc 937 Leu Pro Thr Thr Leu Ile Val Val Ile Ser Trp Val SerPhe Trp Leu 255 260 265 gac gta gac gcc ata ccc gcc cgt gtc acc ctg ggcgta acc acg ctg 985 Asp Val Asp Ala Ile Pro Ala Arg Val Thr Leu Gly ValThr Thr Leu 270 275 280 285 ctc acc atc tca tcc aag ggt gcc ggt atc caggga aac ctg cct ccc 1033 Leu Thr Ile Ser Ser Lys Gly Ala Gly Ile Gln GlyAsn Leu Pro Pro 290 295 300 gtc tcg tac atc aag gcc atg gac gtc tgg atagga tcc tgt act tcg 1081 Val Ser Tyr Ile Lys Ala Met Asp Val Trp Ile GlySer Cys Thr Ser 305 310 315 ttt gtc ttt gcg gcc ctt cta gag ttc aca ttcgtc aac tat ctc tgg 1129 Phe Val Phe Ala Ala Leu Leu Glu Phe Thr Phe ValAsn Tyr Leu Trp 320 325 330 agg cgg ctg ccc aat aag cgc cca tct tct gacgta ccg gtg acg gat 1177 Arg Arg Leu Pro Asn Lys Arg Pro Ser Ser Asp ValPro Val Thr Asp 335 340 345 ata cca agc gac ggc tca aag cat gac att gcggca cag ctc gta ctc 1225 Ile Pro Ser Asp Gly Ser Lys His Asp Ile Ala AlaGln Leu Val Leu 350 355 360 365 gac aag aat gga cac acc gaa gtt cgc acgttg gtc caa gcg atg cca 1273 Asp Lys Asn Gly His Thr Glu Val Arg Thr LeuVal Gln Ala Met Pro 370 375 380 cgc agc gtc gga aaa gtg aag gcc aag cagatt gat caa ctc agc cga 1321 Arg Ser Val Gly Lys Val Lys Ala Lys Gln IleAsp Gln Leu Ser Arg 385 390 395 gtc gcc ttt ccc gct ctt ttt ctc ctc ttcaac ctc gtg tac tgg ccg 1369 Val Ala Phe Pro Ala Leu Phe Leu Leu Phe AsnLeu Val Tyr Trp Pro 400 405 410 tac tac att aag tca t aaagaacgta gttttct1402 Tyr Tyr Ile Lys Ser 415 8 418 PRT Rhipicephalus sanguineus 8 MetPhe Gly Val Pro Cys Ser Arg Ala Cys Arg Leu Val Val Val Ile 1 5 10 15Ala Ala Phe Cys Trp Pro Pro Ala Leu Pro Leu Val Pro Gly Gly Val 20 25 30Ser Ser Arg Ala Asn Asp Leu Asp Ile Leu Asp Glu Leu Leu Lys Asn 35 40 45Tyr Asp Arg Arg Ala Leu Pro Ser Ser His Leu Gly Asn Ala Thr Ile 50 55 60Val Ser Cys Glu Ile Tyr Ile Arg Ser Phe Gly Ser Ile Asn Pro Ser 65 70 7580 Asn Met Asp Tyr Glu Val Asp Leu Tyr Phe Arg Gln Ser Trp Leu Asp 85 9095 Glu Arg Leu Arg Lys Ser Thr Leu Ser Arg Pro Leu Asp Leu Asn Asp 100105 110 Pro Lys Leu Val Gln Met Ile Trp Lys Pro Glu Val Phe Phe Ala Asn115 120 125 Ala Lys His Ala Glu Phe Gln Tyr Val Thr Val Pro Asn Val LeuVal 130 135 140 Arg Ile Asn Pro Thr Gly Ile Ile Leu Tyr Met Leu Arg LeuLys Leu 145 150 155 160 Arg Phe Ser Cys Met Met Asp Leu Tyr Arg Tyr ProMet Asp Ser Gln 165 170 175 Val Cys Ser Ile Glu Ile Ala Ser Phe Ser LysThr Thr Glu Glu Leu 180 185 190 Leu Leu Lys Trp Ser Glu Ser Gln Pro ValVal Leu Phe Asp Asn Leu 195 200 205 Lys Leu Pro Gln Phe Glu Ile Glu LysVal Asn Thr Ser Leu Cys Lys 210 215 220 Glu Lys Phe His Ile Gly Glu TyrSer Cys Leu Lys Ala Asp Phe Tyr 225 230 235 240 Leu Gln Arg Ser Leu GlyTyr His Met Val Gln Thr Tyr Leu Pro Thr 245 250 255 Thr Leu Ile Val ValIle Ser Trp Val Ser Phe Trp Leu Asp Val Asp 260 265 270 Ala Ile Pro AlaArg Val Thr Leu Gly Val Thr Thr Leu Leu Thr Ile 275 280 285 Ser Ser LysGly Ala Gly Ile Gln Gly Asn Leu Pro Pro Val Ser Tyr 290 295 300 Ile LysAla Met Asp Val Trp Ile Gly Ser Cys Thr Ser Phe Val Phe 305 310 315 320Ala Ala Leu Leu Glu Phe Thr Phe Val Asn Tyr Leu Trp Arg Arg Leu 325 330335 Pro Asn Lys Arg Pro Ser Ser Asp Val Pro Val Thr Asp Ile Pro Ser 340345 350 Asp Gly Ser Lys His Asp Ile Ala Ala Gln Leu Val Leu Asp Lys Asn355 360 365 Gly His Thr Glu Val Arg Thr Leu Val Gln Ala Met Pro Arg SerVal 370 375 380 Gly Lys Val Lys Ala Lys Gln Ile Asp Gln Leu Ser Arg ValAla Phe 385 390 395 400 Pro Ala Leu Phe Leu Leu Phe Asn Leu Val Tyr TrpPro Tyr Tyr Ile 405 410 415 Lys Ser 9 27 DNA Artificial Sequenceoligonucleotide 9 ggatkccnga ynynttyttn nmnamyg 27 10 24 DNA ArtificialSequence oligonucleotide 10 cnarmarngc ncmgaanayr aayg 24 11 26 DNAArtificial Sequence oligonucleotide 11 canrcnccnr kccanacrtc naynrc 2612 248 DNA Drosophila melanogaster 12 attacttaat acaaatttat ataccatgctgtatgttggt cattgtatca tgggtatcat 60 tctggctgga tcaaggagca gtaccggcgcgagtgtcact gggtgtcacc accctgctga 120 ccatggccac ccagacgtcg ggcataaacgcctccctgcc gcccgtttcc tatacgaagg 180 ccatcgatgt gtggacaggc gtgtgtctgacgttcgtgtt cggggccctg ctcgagttcg 240 ccctggtg 248

What is claimed is:
 1. A purified nucleic acid molecule encoding a R.sanguineus GluCl channel protein, wherein said nucleic acid moleculecomprises: (a) a nucleic acid molecule which encodes an animo acidsequence selected from the group consisting of SEQ ID NOs 2, 4, 6 and 8;(b) a nucleic acid molecule which hybridizes under conditions ofmoderate to high stringency to the complement of a second nucleic acidmolecule which encodes SEQ ID NOs 2, 4, 6 and 8; or, (c) a nucleic acidmolecule which hybridizes under conditions of moderate stringency to thecomplement of a second nucleic acid molecule as set forth in SEQ ID NOs1, 3, 5 and 7; wherein said nucleic acid molecule has at least about a55% identity to at least one of the second nucleic acid molecules as setforth in SEQ ID NOs 1, 3, 5 and
 7. 2. A purified DNA molecule encoding aR. sanguineus GluCl1 channel protein, wherein said protein comprises anamino acid sequence as set forth in SEQ ID NO:2:
 3. An expression vectorfor expressing a R. sanguineus GluCl1 channel protein in a recombinanthost cell wherein said expression vector comprises a DNA molecule ofclaim
 2. 4. A host cell which expresses a recombinant R. sanguineusGluCl1 channel protein wherein said host cell contains the expressionvector of claim
 3. 5. A process for expressing a R. sanguineus GluCl1channel protein in a recombinant host cell, comprising: (a) transfectingthe expression vector of claim 4 into a suitable host cell; and, (b)culturing the host cells of step (a) under conditions which allowexpression of said R. sanguineus GluCl1 channel protein from saidexpression vector.
 6. A purified DNA molecule encoding a R. sanguineusGluCl1 channel protein which consists of a nucleotide sequence as setforth in SEQ ID NO:1.
 7. The DNA molecule of claim 6 which consists ofthe nucleotide sequence from about nucleotide 331 to about nucleotide1683.
 8. A purified DNA molecule encoding a R. sanguineus GluCl1 channelprotein, wherein said protein comprises an amino acid sequence as setforth in SEQ ID NO:4.
 9. An expression vector for expressing a R.sanguineus GluCl1 channel protein in a recombinant host cell whereinsaid expression vector comprises a DNA molecule of claim
 8. 10. A hostcell which expresses a recombinant R. sanguineus GluCl1 channel proteinwherein said host cell contains the expression vector of claim
 9. 11. Aprocess for expressing a R. sanguineus GluCl1 channel protein in arecombinant host cell, comprising: (a) transfecting the expressionvector of claim 10 into a suitable host cell; and, (b) culturing thehost cells of step (a) under conditions which allow expression of saidR. sanguineus GluCl1 channel protein from said expression vector.
 12. Apurified DNA molecule encoding a R. sanguineus GluCl1 channel proteinwhich consists of a nucleotide sequence as set forth in SEQ ID NO:3. 13.The DNA molecule of claim 12 which consists of the nucleotide sequencefrom about nucleotide 502 to about nucleotide
 1854. 14. A purified DNAmolecule encoding a R. sanguineus GluCl1 channel protein, wherein saidprotein comprises an amino acid sequence as set forth in SEQ ID NO:6.15. An expression vector for expressing a R. sanguineus GluCl1 channelprotein in a recombinant host cell wherein said expression vectorcomprises a DNA molecule of claim
 14. 16. A host cell which expresses arecombinant R. sanguineus GluCl1 channel protein wherein said host cellcontains the expression vector of claim
 15. 17. A process for expressinga R. sanguineus GluCl1 channel protein in a recombinant host cell,comprising: (a) transfecting the expression vector of claim 16 into asuitable host cell; and, (b) culturing the host cells of step (a) underconditions which allow expression of said R. sanguineus GluCl1 channelprotein from said expression vector.
 18. A purified DNA moleculeencoding a R. sanguineus GluCl1 channel protein which consists of anucleotide sequence as set forth in SEQ ID NO:5.
 19. The DNA molecule ofclaim 18 which consists of the nucleotide sequence from about nucleotide617 to about nucleotide
 2170. 20. A purified DNA molecule encoding a R.sanguineus GluCl2 channel protein, wherein said protein comprises anamino acid sequence as set forth in SEQ ID NO:8.
 21. An expressionvector for expressing a R. sanguineus GluCl2 channel protein in arecombinant host cell wherein said expression vector comprises a DNAmolecule of claim
 20. 22. A host cell which expresses a recombinant R.sanguineus GluCl2 channel protein wherein said host cell contains theexpression vector of claim
 21. 23. A process for expressing a R.sanguineus GluCl2 channel protein in a recombinant host cell,comprising: (a) transfecting the expression vector of claim 21 into asuitable host cell; and, (b) culturing the host cells of step (a) underconditions which allow expression of said R. sanguineus GluCl2 channelprotein from said expression vector.
 24. A purified DNA moleculeencoding a R. sanguineus GluCl2 channel protein which consists of anucleotide sequence as set forth in SEQ ID NO:7.
 25. The DNA molecule ofclaim 24 which consists of the nucleotide sequence from about nucleotide131 to about nucleotide 1387 .
 26. A R. sanguineus GluCl1 channelprotein substantially free from other proteins which comprises an aminoacid sequence as set forth in SEQ ID NO:2.
 27. A R. sanguineus GluCl1channel protein of claim 26 which is a product of a DNA expressionvector contained within a recombinant host cell.
 28. A substantiallypure membrane preparation comprising the R. sanguineus GluCl1 channelprotein purified from the recombinant host cell of claim
 27. 29. A R.sanguineus GluCl1 channel protein substantially free from other proteinswhich comprises an amino acid sequence as set forth in SEQ ID NO:4. 30.A R. sanguineus GluCl1 channel protein of claim 29 which is a product ofa DNA expression vector contained within a recombinant host cell.
 31. Asubstantially pure membrane preparation comprising the R. sanguineusGluCl1 channel protein purified from the recombinant host cell of claim20.
 32. A R. sanguineus GluCl1 channel protein substantially free fromother proteins which comprises an amino acid sequence as set forth inSEQ ID NO:6.
 33. A R. sanguineus GluCl1 channel protein of claim 32which is a product of a DNA expression vector contained within arecombinant host cell.
 34. A substantially pure membrane preparationcomprising the R. sanguineus GluCl1 channel protein purified from therecombinant host cell of claim
 33. 35. A R. sanguineus GluCl2 channelprotein substantially free from other proteins which comprises an aminoacid sequence as set forth in SEQ ID NO:8.
 36. A R. sanguineus GluCl2channel protein of claim 35 which is a product of a DNA expressionvector contained within a recombinant host cell.
 37. A substantiallypure membrane preparation comprising the R. sanguineus GluCl2 channelprotein purified from the recombinant host cell of claim
 36. 38. A R.sanguineus GluCl1 channel protein which consists of an amino acidsequence selected from the group consisting of amino acid sequences asset forth in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
 39. A R.sanguineus GluCl2 channel protein which consists of an amino acidsequence as set forth in SEQ ID NO:8.
 40. A method of identifying amodulator of a GluCl channel protein, comprising: (a) contacting a testcompound with a R. sanguineus GluCl channel protein selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6; and SEQ IDNO:8; and, (b) measuring the effect of the test compound on the GluClchannel protein.
 41. The method of claim 40 wherein the R. sanguineusGluCl protein of step (a) is a product of a DNA expression vectorcontained within a recombinant host cell.
 42. A method of identifying acompound that modulates glutamate-gated channel protein activity, whichcomprises: a) injecting into a host cell solution a population ofnucleic acid molecules, at least of portion of which encodes a R.sanguineus GluCl channel protein selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8 such thatexpression of said portion of nucleic acid molecules results in anactive glutamate-gated channel; b) adding a test compound into saidsolution; and, c) measuring host cell membrane current at a holdingpotential more positive than the reversal potential for chloride. 43.The method of claim 42 wherein said nucleic acid molecule is selectedfrom the group consisting of complementary DNA, poly A⁺ messenger RNAand complementary RNA.